Publications

Peer Reviewed Publications

2018

Pamenter ME, Lau GY, Richards JG and Milsom Wk (2018). Naked mole rat brain mitochondria electron transport system flux and H+ leak are reduced during acute hypoxia. Journal of Experimental Biology. In press.

Abstract:

Mitochondrial respiration and ATP production are compromised by hypoxia. Naked mole rats (NMRs) are among the most hypoxia-tolerant mammals and reduce metabolic rate in hypoxic environments; however, little is known regarding mitochondrial function during in vivo hypoxia exposure in this species. To address this knowledge gap, we asked whether the function of NMR brain mitochondria exhibits metabolic plasticity during acute hypoxia. Respirometry was utilized to assess whole-animal oxygen consumption rates and high-resolution respirometry and was utilized to assess electron transport system (ETS) function in saponin-permeabilized NMR brain. We found that NMR whole animal oxygen consumption rate reversibly decreased by ~ 85% in acute hypoxia (4 hrs at 3% O2). Similarly, relative to untreated controls, permeabilized brain respiratory flux through the ETS was decreased by ~ 90% in acutely hypoxic animals. Relative to FCCP-uncoupled total ETS flux, this functional decrease was observed equally across all components of the ETS except for complex IV (cytochrome c oxidase), at which flux was further reduced, supporting a regulatory role for this enzyme during acute hypoxia. The maximum enzymatic capacities of ETS complexes I-V were not altered by acute hypoxia; however, the mitochondrial H+-gradient decreased in step with the decrease in ETS respiration. Taken together, our results indicate that NMR brain ETS flux and H+ leak are reduced in a balanced and regulated fashion during acute hypoxia. Changes in NMR mitochondrial metabolic plasticity mirror whole animal metabolic responses to hypoxia.

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Pamenter ME, Lau GY and Richards JG (2018). Effects of cold on brain mitochondrial function . PLoS One. In press.

Abstract:

Therapeutic hypothermia is a strategy that reduces metabolic rate and brain damage during clinically-relevant hypoxic events. Mitochondrial respiration is compromised by hypoxia, with deleterious consequences for the mammalian brain; however, little is known about the effects of reduced temperature on mitochondrial metabolism. Therefore, we examined how mitochondrial function is impacted by temperature using high resolution respirometry to assess electron transport system (ETS) function in saponin-permeabilized mouse brain at 28 and 37ºC. Respirometric analysis revealed that, at the colder temperature, ETS respiratory flux was ~ 40-75% lower relative to at the physiological temperature in all respiratory states and for all fuel substrates tested. In whole brain tissue, the enzyme maximum respiratory rates for complexes I-V were similarly reduced by between 37-88%. Complexes II and V were particularly temperature-sensitive; a temperature-mediated decrease in complex II activity may support a switch to complex I mediated ATP-production, which is considerably more oxygen-efficient. Finally, the mitochondrial H+-gradient was more tightly coupled, indicating that mitochondrial respiration is more efficient at the colder temperature. Taken together, our results suggest that improvements in mitochondrial function with colder temperatures may contribute to energy conservation and enhance cellular viability in hypoxic brain.

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Branigan T Elkhalifa S and Pamenter ME (2018). Behavioural Responses to Environmental Hypercapnia in Two Eusocial Species of African Mole Rats. Journal of Comparative Physiology A.204(9-10):811-819.

Abstract:

Damaraland and naked mole rat are eusocial mammals that live in crowded burrows in which CO2 is elevated. These species are thought to be highly tolerant of CO2 but their behavioural responses to hypercapnia are poorly understood. We hypothesized that Damaraland and naked mole rats would exhibit blunted behavioural responses to hypercapnia and predicted that their activity levels would be unaffected at low to moderate (2-5%) CO2 but increased at > 7% CO2. To test this, we exposed Damaraland and naked mole rats to stepwise increases in environmental CO2 (0-10%) and measured activity, exploratory behaviour, and body temperature. Surprisingly, we found that both species exhibited no differences in movement velocity, distance travelled, zone transitions (exploration), or body temperature at any level of environmental hypercapnia. Conversely, when carbonic anhydrase was inhibited with acetazolamide (50 mg•kg-1 intraperitonially, to increase whole animal acidosis), exploration was significantly elevated relative to hypercapnic controls in both species at all levels of inhaled CO2, and naked mole rat body temperature decreased in > 7% CO2. We conclude that both species are largely non-responsive to environmental CO2, and that this tolerance may be dependent on bicarbonate buffering at the level of the kidney or within the blood.

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Kirby AM Fairman GD and Pamenter ME (2018). Atypical behavioural, metabolic, and thermoregulatory responses to hypoxia in the naked mole rat (Heterocephalus glaber). Journal of Zoology. 305:106-115.

Abstract:

Hypoxia compromises aerobic energy production at the cellular level but hypoxic environments are commonly encountered by mammals. In response, mammals typically exhibit compensatory physiological and behavioural adaptations that help to restore energetic homeostasis by reducing physical activity and body temperature (Tb) to lower metabolic demand. Naked mole rats (NMRs) are among the most hypoxia-tolerant mammals identified but their behavioural and thermal responses to acute hypoxia are poorly characterized. Using behavioural tracking software, we examined the effects of acute hypoxia (1 hr at 7% O2) on physical activity and Tb in animals held at their natural burrow temperature (30˚C), or at temperatures above (38˚C) or below (20˚C). In separate experiments, we used respirometry to measure metabolic rate under the same conditions. Physical activity decreased ~ 26-60% and Tb decreased by ~ 1.9-3.7˚C during hypoxia in all temperatures. Normoxic metabolic rate was highest at 20˚C, but was suppressed to similar rates in hypoxia across all temperatures. When animals were given the opportunity to escape their burrow temperature to either warmer or colder chambers during the hypoxic episode, NMRs surprisingly avoided the cold chamber¬, suggesting they are unable to take advantage of anapyrexia. Conversely, NMRs were able to use behavioural thermoregulation to maintain Tb¬ in hypoxia when given the choice of temperatures within and above their burrow temperature. Taken together, these findings indicate that NMRs respond to acute hypoxia with reversible decreases in physical activity, Tb¬, and metabolic rate across a range of ambient temperatures to reduce oxygen requirements.

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Buck LT and Pamenter ME (2018). The Hypoxia-Tolerant Vertebrate Brain: Arresting Synaptic Activity. Comparative Biochemistry and Physiology B. Invited contribution, special issues: Tribute to Peter W Hochachka. 224:61-70.

Abstract:

The ion channel arrest hypothesis has been the foundation of three decades of research into the underlying mechanisms of hypoxia/anoxia tolerance in several key species, including: painted turtles, goldfish, crucian carp, naked mole rats, and arctic and ground squirrels. The hypothesis originally stated that hypoxia/anoxia tolerant species ought to have fewer ion channels per area membrane and/or mechanisms to regulate the permeability of ions through channels. Today we can add to this and include mechanisms to remove channels from membranes and the expression of low conductance isoforms. Furthermore, possible oxygen sensing mechanisms in brain include a link to mitochondrial function, changes in the concentration of intracellular Ca2+ and reactive oxygen species, and activation of protein kinase C and a phosphatase. Importantly ion channel arrest leads to a decrease in metabolic rate that is fundamental to survival without oxygen and in brain is reflected in decreased action potential frequency or spike arrest. This results not only from a decrease in excitatory glutamatergic receptor currents but also by an increase in inhibitory GABAergic receptor currents. The surprising finding that ionic conductance through some ion channels increases is novel and contrary to the ion channel arrest hypothesis. The major insight that this offers is that key regulatory events are occurring at the level of the synapse and we therefore propose the “synaptic arrest hypothesis”.

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Houlahan CR, Kirby AM, Dzal YA, Fairman GD and Pamenter ME (2018). Divergent behavioural responses to acute hypoxia between individuals and groups of naked mole rats . Comparative Biochemistry and Physiology B. Invited contribution, special issues: Tribute to Peter W Hochachka. 224:38-44.

Abstract:

Most small rodents reduce energy demand in hypoxia via behavioural strategies. For example, animals may reduce their activity, and/or move to colder environments or alter huddling strategies to take advantage of anapyretical energy savings. Naked mole rats (NMRs) are among the most hypoxia tolerant mammals and are highly social; social interactions also have a significant impact on behaviour. Therefore, this species offers a fascinating model in which to study trade-offs between social interactions and energy conservation in hypoxia. We hypothesized that the need to conserve energy in hypoxia supersedes the impetus of sociality in this species and predicted that, in hypoxia, behaviour would not differ between individuals or groups of NMRs. To test this hypothesis, we placed awake, freely behaving NMRs, alone or in groups of 2 or 4, into a temperature-controlled apparatus and measured behavioural activity during 1 hr each of normoxia (21% O2), acute hypoxia (7% O2), and normoxic recovery. We found that in normoxia, groups of 4 NMRs were significantly more active in all temperatures than were groups of 1-2 NMRs. When exposed to hypoxia, individual NMRs were ~ 50% less active and their speed was reduced relative to normoxic levels. Conversely, groups of 2 or 4 NMRs exhibited minor or insignificant decreases in time spent active and speed in hypoxia and huddling behaviour was not altered. Our findings suggest that social interactions influence behavioural strategies employed by NMRs in hypoxia.

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2017

Ilacqua AN, Kirby AM, and Pamenter ME (2017). Behavioural responses of naked mole rats to acute hypoxia and anoxia. Biology letters. 13(12):20170545.

Abstract:

Naked mole rats (NMRs) are among the most hypoxia-tolerant mammals. Other species respond to hypoxia by either escaping the hypoxic environment or drastically decreasing behavioural activity and body temperature (Tb) to conserve energy. However, NMRs rarely leave their underground burrows, which are putatively hypoxic and thermally stable near the NMRs’ preferred Tb. Therefore, we asked whether NMRs are able to employ behavioural and thermoregulatory strategies in response to hypoxia despite their need to remain active and the minimal thermal scope in their burrows. We exposed NMRs to progressively deeper levels of hypoxia (from 21 to 0% O2) while measuring their behaviour and Tb. Behavioural activity decreased 40-60% in hypoxia and Tb decreased slightly in moderate hypoxia (5-9%) and then further with deeper hypoxia (3% O2). However, even at 3% O2 NMRs remained somewhat active and warm, and continued to explore their environment. Remarkably, NMRs were active for > 90 secs in acute anoxia and Tb and metabolic rate decreased rapidly. We conclude that NMRs are adapted to remain awake and functional even at the extremes of their hypoxia-tolerance. This adaptation likely reflects variable and challenging levels of environmental hypoxia in the natural habitat of this species.

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2016

Chung D, Dzal YA, Seow A, Milsom WK and Pamenter ME (2016). Naked mole rats exhibit metabolic but not ventilatory plasticity following chronic sustained hypoxia. Proceedings of the Royal Society B. 283(1827):20162016.

Abstract:

Naked mole rats are among the most hypoxia-tolerant mammals identified and live in chronic hypoxia throughout their lives. The mechanisms underlying this tolerance, however, are poorly understood. Most vertebrates hyperventilate in acute hypoxia and exhibit an enhanced hyperventilation following acclimatization to chronic sustained hypoxia (CSH). Conversely, naked mole rats do not hyperventilate in acute hypoxia and their response to CSH has not been examined. In this study we explored mechanisms of plasticity in the control of the hypoxic ventilatory response (HVR) and hypoxic metabolic response (HMR) of freely behaving naked mole rats following 8-10 days of chronic sustained normoxia (CSN) or CSH. Specifically, we investigated the role of the major inhibitory neurotransmitter γ-amino butyric acid (GABA) in mediating these responses. Our study yielded three important findings. First, naked mole rats did not exhibit ventilatory plasticity following CSH, which is unique among adult animals studied to date. Second, GABA receptor (GABAR) antagonism altered breathing patterns in CSN and CSH animals and modulated the acute HVR in CSN animals. Third, naked mole rats exhibited GABAR-dependent metabolic plasticity following long-term hypoxia, such that the basal metabolic rate was ~25% higher in normoxic CSH animals than CSN animals, and GABAR antagonists modulated this increase.

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Pamenter ME and Powell FL (2016). Time domains of the hypoxic ventilatory response and their molecular basis. Comprehensive Physiology. 6(3):1345-1385.

Abstract:

Ventilatory responses to hypoxia vary widely depending on the pattern and length of hypoxic exposure. Acute, prolonged, or intermittent hypoxic episodes can increase or decrease breathing for seconds to years, both during the hypoxic stimulus, and also after its removal. These myriad effects are the result of a complicated web of molecular interactions that underlie plasticity in the respiratory control reflex circuits and ultimately control the physiology of breathing in hypoxia. Since the time domains of the physiological hypoxic ventilatory response (HVR) were identified, considerable research effort has gone towards elucidating the underlying molecular mechanisms that mediate these varied responses. This research has begun to describe complicated and plastic interactions in the relay circuits between the peripheral chemoreceptors and the ventilatory control circuits within the central nervous system. Intriguingly, many of these molecular pathways seem to share key components between the different time domains, suggesting that varied physiological HVRs are the result of specific modifications to overlapping pathways. This review highlights what has been discovered regarding the cell and molecular level control of the time domains of the HVR, and highlights key areas where further research is required. Understanding the molecular control of ventilation in hypoxia has important implications for basic physiology and is emerging as an important component of several clinical fields.

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Pamenter ME (2016). Comparative insights into mitochondrial adaptations to anoxia in brain. Neural Regeneration Research. 11(5):723-724.

Pamenter ME, Gomez CR, Richards JG and Milsom WK (2016). Mitochondrial responses to prolonged anoxia in brain of red-eared slider turtles. Biology Letters. 12:20150797.

Abstract:

Mitochondria are central to aerobic energy production and play a key role in neuronal signalling. During anoxia, however, the mitochondria of most vertebrates initiate deleterious cell death cascades. Nonetheless, a handful of vertebrate species, including some freshwater turtles, are remarkably tolerant of low oxygen environments and survive months of anoxia without apparent damage to brain tissue. This tolerance suggests that mitochondria in the brains of such species are adapted to withstand prolonged anoxia, but little is known about potential neuroprotective responses. In this study, we address such mechanisms by comparing mitochondrial function between brain tissues isolated from cold-acclimated red-eared slider turtles (Trachemys scripta elegans) exposed to two weeks of either normoxia or anoxia. We found that brain mitochondria from anoxia-acclimated turtles exhibited a unique phenotype of remodelling relative to normoxic controls, including: (i) decreased citrate synthase and F1FO-ATPase activity but maintained protein content, (ii) markedly reduced aerobic capacity, and (iii) mild uncoupling of the mitochondrial proton gradient. These data suggest that turtle brain mitochondria respond to low oxygen stress with a unique suite of changes tailored towards neuroprotection.

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2015

Hogg DW, Pamenter ME, Dukoff DJ and Buck LT (2015). Decreases in mitochondrial reactive oxygen species initiate GABAA receptor-mediated electrical suppression in anoxia-tolerant turtle neurons. Journal of Physiology (London). 593(10):2311-2326.

Abstract:

Anoxia induces hyper-excitability and cell death in mammalian brain but in the anoxia-tolerant western painted turtle (Chrysemys picta bellii) neuronal electrical activity is suppressed (i.e. spike arrest), adenosine triphosphate (ATP) consumption is reduced, and cell death does not occur. Electrical suppression is primarily the result of enhanced γ-aminobutyric acid (GABA) transmission; however, the underlying mechanism responsible for initiating oxygen-sensitive GABAergic spike arrest is unknown. In turtle cortical pyramidal neurons there are three types of GABAA receptor-mediated currents: spontaneous inhibitory postsynaptic currents (IPSCs), giant IPSCs and tonic currents. The aim of this study was to assess the effects of reactive oxygen species (ROS) scavenging on these three currents since ROS levels naturally decrease with anoxia and may serve as a redox signal to initiate spike arrest. We found that anoxia, pharmacological ROS scavenging, or inhibition of mitochondrial ROS generation enhanced all three types of GABA currents, with tonic currents comprising #50% of the total current. Application of hydrogen peroxide inhibited all three GABA currents, demonstrating a reversible redox-sensitive signalling mechanism. We conclude that anoxia-mediated decreases in mitochondrial ROS production are sufficient to initiate a redox-sensitive inhibitory GABA signalling cascade that suppresses electrical activity when oxygen is limited. This unique strategy for reducing neuronal ATP consumption during anoxia represents a natural mechanism in which to explore therapies to protect mammalian brain from low-oxygen insults.

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Dzal YA, Jenkin SE, Lague SL, Reichert MN, York JM and Pamenter ME, (2015). From chemosensory cell to life history: how oxygen has influenced the evolution of vertebrate physiology. Comparative Biochemistry and Physiology A. Invited contribution, special issue: Tribute to William Milsom. 186:4-26.

Abstract:

In response to varying environmental and physiological challenges, vertebrates have evolved complex and often overlapping systems. These systems detect changes in environmental oxygen availability and respond by increasing oxygen supply to the tissues and/or by decreasing oxygen demand at the cellular level. This suite of responses is termed the oxygen transport cascade and is comprised of several components. These components include 1) chemosensory detectors that sense changes in oxygen, carbon dioxide, and pH in the blood, and initiate changes in 2) ventilation and 3) cardiac work, thereby altering the rate of oxygen delivery to, and carbon dioxide clearance from, the tissues. In addition, changes in 4) cellular and systemic metabolism alters tissue-level metabolic demand. Thus the need for oxygen can be managed locally when increasing oxygen supply is not sufficient or possible. Together, these mechanisms provide a spectrum of responses that facilitate the maintenance of systemic oxygen homeostasis in the face of environmental hypoxia or physiological oxygen depletion (i.e. due to exercise or disease). Bill Milsom has dedicated his career to the study of these responses across phylogenies, repeatedly demonstrating the power of applying the comparative approach to physiological questions. The focus of this review is to discuss the anatomy, signalling pathways, and mechanics of each step of the oxygen transport cascade from the perspective of a Milsomite. That is, by taking into account the developmental, physiological, and evolutionary components of questions related to oxygen transport. We also highlight examples of some of the remarkable species that have captured Bill’s attention through their unique adaptations in multiple components of the oxygen transport cascade, which allow them to achieve astounding physiological feats. Bill’s research examining the oxygen transport cascade has provided important insight and leadership to the study of the diverse suite of adaptations that maintain cellular oxygen content across vertebrate taxa, which underscores the value of the comparative approach to the study of physiological systems.

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Pamenter ME, Go, A, Fu Z and Powell FL (2015). Ventilatory acclimatization to hypoxia is not mediated by neuronal nitric oxide synthase in the NTS of rat. Journal of Applied Physiology. 118(6):750-759.

Abstract:

When exposed to a hypoxic environment, the body’s first response is a reflex increase in ventilation, termed the hypoxic ventilatory response (HVR). With chronic sustained hypoxia (CSH), such as during acclimatization to high altitude, an additional time-dependent increase in ventilation occurs, which increases the HVR and is termed ventilatory acclimatization to hypoxia (VAH). This secondary increase persists after exposure to CSH and involves plasticity within the circuits in the central nervous system that control breathing. The mechanisms of HVR plasticity are currently poorly understood. We hypothesized that changes in neuronal nitric oxide synthase (nNOS) activity or expression in the nucleus tractus solitarius contribute to this plasticity and underlie VAH in rats. To test this, we treated rats held in normoxia or 10% O2 (CSH, PIO2 = 70 Torr) for 7-9 days and measured ventilation in conscious, unrestrained animals before and after microinjecting the general NOS antagonist L-NG-Nitroarginine methyl ester into the nucleus tractus solitarius (NTS) or systemically injecting the nNOS-specific antagonist S-methyl-l-thiocitrulline. Localization of injection sites in the NTS was confirmed by histology following the experiment. We found that 1) neither NTS-specific nor systemic nNOS antagonism had any effect on hypoxia-mediated changes in breathing or metabolism (P > 0.05), but 2) nNOS protein expression was increased in the middle and caudal NTS by CSH. A persistent HVR after nNOS blockade in the NTS contrasts with results in awake mice, and our findings do not support the hypotheses that nNOS in the NTS contribute to the HVR or VAH in awake rats.

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Pamenter ME, Dzal Y and Milsom WK (2015). Adenosine receptors inhibit the hypoxic ventilatory response but not the hypoxic metabolic response in the naked mole rat during acute hypoxia. Proceedings of the Royal Society B. 282(1800):20141722.

Abstract:

Naked mole rats are the most hypoxia-tolerant mammals identified; however, the mechanisms underlying this tolerance are poorly understood. Using whole-animal plethysmography and open-flow respirometry, we examined the hypoxic metabolic response (HMR), hypoxic ventilatory response (HVR) and hypoxic thermal response in awake, freely behaving naked mole rats exposed to 7% O₂ for 1 h. Metabolic rate and ventilation each reversibly decreased 70% in hypoxia (from 39.6 ± 2.9 to 12.1 ± 0.3 ml O₂ min(-1) kg(-1), and 1412 ± 244 to 417 ± 62 ml min(-1) kg(-1), respectively; p < 0.05), whereas body temperature was unchanged and animals remained awake and active. Subcutaneous injection of the general adenosine receptor antagonist aminophylline (AMP; 100 mg kg(-1), in saline), but not control saline injections, prevented the HVR but had no effect on the HMR. As a result, AMP-treated naked mole rats exhibited extreme hyperventilation in hypoxia. These animals were also less tolerant to hypoxia, and in some cases hypoxia was lethal following AMP injection. We conclude that in naked mole rats (i) hypoxia tolerance is partially dependent on profound hypoxic metabolic and ventilatory responses, which are equal in magnitude but occur independently of thermal changes in hypoxia, and (ii) adenosine receptors mediate the HVR but not the HMR.

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Pamenter ME, and Haddad GG (2015). High-throughput cell death assays. Methods in Molecular Biology. Invited contribution. 1254:153-163.

Abstract:

High-throughput screens (HTS) are powerful tools that permit the rapid evaluation of thousands of samples in a cost-effective manner and minimize sample and reagent consumption. Such assays have recently begun to be utilized to evaluate cell death modalities and also the cytoprotective efficacy of compounds against a wide variety of stresses. Here we describe the design, preparation, and undertaking of HTS-appropriate assays that utilize simple and cost-effective fluorophore- and luminescence-based functional readouts of cell viability. These assays permit the examination of 96–384 compounds in a single multiwell plate with highly robust statistical significance at a fraction of the financial and work cost of traditional approaches.

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2014

Pamenter ME, Nguyen J, Carr JA and Powell FL (2014). The effect of combined glutamate receptor blockade in the NTS on the hypoxic ventilatory responses in awake rats differs from the effect of individual glutamate receptor blockade. Physiological Reports. 2(7):e12092.

Abstract:

Ventilatory acclimatization to hypoxia (VAH) increases the hypoxic ventilatory response (HVR) and causes persistent hyperventilation when normoxia is restored, which is consistent with the occurrence of synaptic plasticity in acclimatized animals. Recently, we demonstrated that antagonism of individual glutamate receptor types (GluRs) within the nucleus tractus solitarii (NTS) modifies this plasticity and VAH (J. Physiol. 592(8):1839–1856); however, the effects of combined GluR antagonism remain unknown in awake rats. To evaluate this, we exposed rats to room air or chronic sustained hypobaric hypoxia (CSH, PIO2 = 70 Torr) for 7–9 days. On the experimental day, we microinjected artificial cerebrospinal fluid (ACSF: sham) and then a “cocktail” of the GluR antagonists MK-801 and DNQX into the NTS. The location of injection sites in the NTS was confirmed by glutamate injections on a day before the experiment and with histology following the experiment. Ventilation was measured in awake, unrestrained rats breathing normoxia or acute hypoxia (10% O2) in 15-min intervals using barometric pressure plethysmography. In control (CON) rats, acute hypoxia increased ventilation; NTS microinjections of GluR antagonists, but not ACSF, significantly decreased ventilation and breathing frequency in acute hypoxia but not normoxia (P < 0.05). CSH increased ventilation in hypoxia and acute normoxia. In CSH-conditioned rats, GluR antagonists in the NTS significantly decreased ventilation in normoxia and breathing frequency in hypoxia. A persistent HVR after combined GluR blockade in the NTS contrasts with the effect of individual GluR blockade and also with results in anesthetized rats. Our findings support the hypotheses that GluRs in the NTS contribute to, but cannot completely explain, VAH in awake rats.

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Pamenter ME, and Haddad GG (2014). Do BK channels mediate glioma hypoxia-tolerance? Channels. Invited contribution. 8(3):1-2.

Introduction:

Like all tumors, human gliomas are remarkably tolerant of hypoxia. Conversely, hypoxia rapidly activates cell death pathways in healthy brain cells. Therapies aimed at reversing the hypoxia-tolerance of cancerous cells offer considerable promise in the treatment of brain tumors; however, the underlying cellular mechanisms that permit tumor cells to tolerate prolonged hypoxia are poorly understood. Key to normal cellular responses to hypoxia are a number of ion channels whose expression or activity are modified by changes in oxygen. Such changes in ion channel function alter cellular ion gradients that control downstream signaling cascades, which in turn mediate a wide variety of intraand inter-cellular 2nd messenger systems that are critical to cellular viability, growth, and proliferation. Therefore, the ability of ion channels to respond to hypoxia and beneficially regulate these downstream cellular pathways plays a key role in determining cellular tolerance to low oxygen stress. As such, differences in the oxygen-sensitivity of ion channels between healthy vs. cancerous cells are excellent candidates to contribute to the hypoxia-tolerance of tumor cells. Of particular interest in the search for treatments of human glioma is one particular family of ion channels, the Ca2+-activated and voltage-dependent K+ (BK) channels, which are found in both the plasmalemmal and mitochondrial inner membranes.

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Pamenter ME, Go A, Fu Z, Carr JA, Reid SG and Powell FL (2014). Glutamate receptors in the nucleus tractus solitarii mediate ventilatory acclimatization to hypoxia in rat. Journal of Physiology (London). 592(8):1839-1856.

Abstract:

When exposed to a hypoxic environment the body’s first response is a reflex increase in ventilation, termed the hypoxic ventilatory response (HVR). With chronic sustained hypoxia (CSH), such as during acclimatization to high altitude, an additional time-dependent increase in ventilation occurs, which increases the HVR. This secondary increase persists after exposure to CSH and involves plasticity within the circuits in the central nervous system that control breathing. Currently these mechanisms of HVR plasticity are unknown and we hypothesized that they involve glutamatergic synapses in the nucleus tractus solitarius (NTS), where afferent endings from arterial chemoreceptors terminate. To test this, we treated rats held in normoxia (CON) or 10% O2 (CSH) for 7 days and measured ventilation in conscious, unrestrained animals before and after microinjecting glutamate receptor agonists and antagonists into the NTS. In normoxia, AMPA increased ventilation 25% and 50% in CON and CSH, respectively, while NMDA doubled ventilation in both groups (P < 0.05). Specific AMPA and NMDA receptor antagonists (NBQX and MK801, respectively) abolished these effects. MK801 significantly decreased the HVR in CON rats, and completely blocked the acute HVR in CSH rats but had no effect on ventilation in normoxia. NBQX decreased ventilation whenever it was increased relative to normoxic controls; i.e. acute hypoxia in CON and CSH, and normoxia in CSH. These results support our hypothesis that glutamate receptors in the NTS contribute to plasticity in the HVR with CSH. The mechanism underlying this synaptic plasticity is probably glutamate receptor modification, as in CSH rats the expression of phosphorylated NR1 and GluR1 proteins in the NTS increased 35% and 70%, respectively, relative to that in CON rats.

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Pamenter ME, (2014). Mitochondria: A multimodal hub of hypoxia-tolerance. Canadian Journal of Zoology. Invited review, special issue: Animal Mitochondria. 92:569-589.

Abstract:

Decreased oxygen availability impairs cellular energy production and, without a coordinated and matched decrease in energy consumption, cellular and whole organism death rapidly ensues. Of particular interest are mechanisms that protect brain from low oxygen injury, as this organ is not only the most sensitive to hypoxia, but must also remain active and functional during low oxygen stress. As a result of natural selective pressures, some species have evolved molecular and physiological mechanisms to tolerate prolonged hypoxia with no apparent detriment. Among these mechanisms are a handful of responses that are essential for hypoxia tolerance, including (i) sensors that detect changes in oxygen availability and initiate protective responses; (ii) mechanisms of energy conservation; (iii) maintenance of basic brain function; and (iv) avoidance of catastrophic cell death cascades. As the study of hypoxia-tolerant brain progresses, it is becoming increasingly apparent that mitochondria play a central role in regulating all of these critical mechanisms. Furthermore, modulation of mitochondrial function to mimic endogenous neuroprotective mechanisms found in hypoxia-tolerant species confers protection against otherwise lethal hypoxic stresses in hypoxia-intolerant organs and organisms. Therefore, lessons gleaned from the investigation of endogenous mecha- nisms of hypoxia tolerance in hypoxia-tolerant organisms may provide insight into clinical pathologies related to low oxygen stress.

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Gu XQ*, Pamenter ME* Siemen D*, Sun X and Haddad GG (2014). O2-sensitive BK channels are not O2-sensitive in hypoxia-tolerant human glioma cells. Glia. 62(4):504-13.

Abstract:

Tumor cells are resistant to hypoxia but the underlying mechanism(s) of this tolerance remain poorly understood. In healthy brain cells, plasmalemmal Ca(2+)-activated K(+) channels ((plasma)BK) function as oxygen sensors and close under hypoxic conditions. Similarly, BK channels in the mitochondrial inner membrane ((mito)BK) are also hypoxia sensitive and regulate reactive oxygen species production and also permeability transition pore formation. Both channel populations are therefore well situated to mediate cellular responses to hypoxia. In tumors, BK channel expression increases with malignancy, suggesting these channels contribute to tumor growth; therefore, we hypothesized that the sensitivity of (plasma)BK and/or (mito)BK to hypoxia differs between glioma and healthy brain cells. To test this, we examined the electrophysiological properties of (plasma)BK and (mito)BK from a human glioma cell line during normoxia and hypoxia. We observed single channel activities in whole cells and isolated mitoplasts with slope conductance of 199 ± 8 and 278 ± 10 pA, respectively. These currents were Ca(2+)- and voltage-dependent, and were inhibited by the BK channel antagonist charybdotoxin (0.1 μM). (plasma)BK could only be activated at membrane potentials >+40 mV and had a low open probability (NPo) that was unchanged by hypoxia. Conversely, (mito)BK were active across a range of membrane potentials (-40 to +40 mV) and their NPo increased during hypoxia. Activating (plasma)BK, but not (mito)BK induced cell death and this effect was enhanced during hypoxia. We conclude that unlike in healthy brain cells, glioma (mito)BK channels, but not (plasma)BK channels are oxygen sensitive.

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2013

Pamenter ME, and Powell FL (2013). Invited review: Signaling mechanisms of long-term facilitation of breathing with intermittent hypoxia. F1000 Reports. 5:23.

Abstract:

Intermittent hypoxia causes long-term facilitation (LTF) of respiratory motor nerve activity and ventilation, which manifests as a persistent increase over the normoxic baseline for an hour or more after the acute hypoxic ventilatory response. LTF is likely involved in sleep apnea, but its exact role is uncertain. Previously, LTF was defined as a serotonergic mechanism, but new evidence shows that multiple signaling pathways can elicit LTF. This raises new questions about the interactions between signaling pathways in different time domains of the hypoxic ventilatory response, which can no longer be defined simply in terms of neurochemical mechanisms.

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Pamenter ME, Perkins GA, Gu XQ, Ellisman MH and Haddad GG (2013). DIDS (4,4’-diisothiocyano-2,2’-stilbenedisulfonic acid) induces apoptotic cell death in cultured hippocampal neurons and is not protective against ischemic stress. PLoS One. 8(4):e60804.

Abstract:

DIDS is a commonly used anion channel antagonist that is putatively cytoprotective against ischemic insult. However, recent reports indicate potentially deleterious secondary effects of DIDS. To assess the impact of DIDS on cellular viability comprehensively we examined neuronal morphology and function through 24 hours treatment with ACSF 6 DIDS (40 or 400 mM). Control cells were unchanged, whereas DIDS induced an apoptotic phenotype (chromatin condensation, nuclear fragmentation and cleavage of the nuclear membrane protein lamin A, expression of pro-apoptotic proteins c-Jun N- terminal kinase 3, caspase 3, and cytochrome C, Annexin V staining, RNA degradation, and oligonucleosomal DNA cleavage). These deleterious effects were mediated by DIDS in a dose- and time-dependant manner, such that higher [DIDS] induced apoptosis more rapidly while apoptosis was observed at lower [DIDS] with prolonged exposure. In an apparent paradox, despite a clear overall apoptotic phenotype, certain hallmarks of apoptosis were not present in DIDS treated cells, including mitochondrial fission and loss of plasma membrane integrity. We conclude that DIDS induces apoptosis in cultured hippocampal neurons, in spite of the fact that some common hallmarks of cell death pathways are prevented. These contradictory effects may cause false-positive results in certain assays and future evaluations of DIDS as a neuroprotective agent should incorporate multiple viability assays.

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2012

Pamenter ME, Perkins GA, McGinness AK, Gu XQ, Ellisman MH and Haddad GG (2012). Autophagy and apoptosis are induced in neurons and astrocytes treated with an in vitro mimic of the ischemic penumbra. PLoS One. 7(12):e51469.

Abstract:

The development of clinical stroke therapies remains elusive. The neuroprotective efficacies of thousands of molecules and compounds have not yet been determined; however, screening large volumes of potential targets in vivo is severely rate limiting. High throughput screens (HTS) may be used to discover promising candidates, but this approach has been hindered by the lack of a simple in vitro model of the ischemic penumbra, a clinically relevant region of stroke-afflicted brain. Recently, our laboratory developed such a mimic (ischemic solution: IS) suitable for HTS, but the etiology of stress pathways activated by this model are poorly understood. The aim of the present study was to determine if the cell death phenotype induced by IS accurately mimics the in vivo penumbra and thus whether our model system is suitable for use in HTS. We treated cultured neuron and astrocyte cell lines with IS for up to 48 hrs and examined cellular energy state ([ATP]), cell and organelle morphology, and gene and molecular profiles related to stress pathways. We found that IS-treated cells exhibited a phenotype of mixed apoptosis/autophagy characteristic of the in vivo penumbra, including: (1) short-term elevation of [ATP] followed by progressive ATP depletion and Poly ADP Ribose Polymerase cleavage, (2) increased vacuole number in the cytoplasm, (3) mitochondrial rupture, decreased mitochondrial and cristae density, release of cytochrome C and apoptosis inducing factor, (4) chromatin condensation, nuclear lamin A and DNA cleavage, fragmentation of the nuclear envelope, and (5) altered expression of mRNA and proteins consistent with autophagy and apoptosis. We conclude that our in vitro model of the ischemic penumbra induces autophagy and apoptosis in cultured neuron and astrocyte cell lines and that this mimic solution is suitable for use in HTS to elucidate neuroprotective candidates against ischemic penumbral cell death.

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Pamenter ME, Hogg DW, Gu XQ, Buck LT and Haddad GG (2012). Painted turtle cortex is resistant to an in vitro mimic of the ischemic mammalian penumbra. Journal of Cerebral Blood Flow and Metabolism. 32(11)2033-2043.

Abstract:

Anoxia or ischemia causes hyperexcitability and cell death in mammalian neurons. Conversely, in painted turtle brain anoxia increases c-amino butyric acid (GABA)ergic suppression of spontaneous electrical activity, and cell death is prevented. To examine ischemia tolerance in turtle neurons, we treated cortical sheets with an in vitro mimic of the penumbral region of stroke- afflicted mammalian brain (ischemic solution, IS). We found that during IS perfusion, neuronal membrane potential (Vm) and the GABAA receptor reversal potential depolarized to a similar steady state (—92±2 to —28±3mV, and —75±1 to —35±3mV, respectively), and whole-cell conductance (Gw) increased > 3-fold (from 4±0.2 to 15±1 nS). These neurons were electrically quiet and changes reversed after reperfusion. GABA receptor antagonism prevented the IS-mediated increase in Gw and neurons exhibited enhanced electrical excitability and rapid and irreversible rundown of Vm during reperfusion. These results suggest that inhibitory GABAergic mechanisms also suppress electrical activity in ischemic cortex. Indeed, after 4 hours of IS treatment neurons did not exhibit any apparent damage; while at 24 hours, only early indicators of apoptosis were present. We conclude that anoxia-tolerant turtle neurons are tolerant of exposure to a mammalian ischemic penumbral mimic solution.

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Pamenter ME, Ryu J, Hua ST, Perkins GA, Mendiola VL, Gu XQ, Ellisman MH and Haddad GG (2012). DIDS prevents ischemic membrane degradation in cultured hippocampal neurons by inhibiting matrix metalloproteinase release. PLoS One. 7(8):e43995.

Abstract:

During stroke, cells in the infarct core exhibit rapid failure of their permeability barriers, which releases ions and inflammatory molecules that are deleterious to nearby tissue (the penumbra). Plasma membrane degradation is key to penumbral spread and is mediated by matrix metalloproteinases (MMPs), which are released via vesicular exocytosis into the extracellular fluid in response to stress. DIDS (4,49-diisothiocyanatostilbene-2,29-disulphonic acid) preserves membrane integrity in neurons challenged with an in vitro ischemic penumbral mimic (ischemic solution: IS) and we asked whether this action was mediated via inhibition of MMP activity. In cultured murine hippocampal neurons challenged with IS, intracellular proMMP-2 and -9 expression increased 4–10 fold and extracellular latent and active MMP isoform expression increased 2–22 fold. MMP-mediated extracellular gelatinolytic activity increased ,20–50 fold, causing detachment of 32.164.5% of cells from the matrix and extensive plasma membrane degradation (.60% of cells took up vital dyes and .60% of plasma membranes were fragmented or blebbed). DIDS abolished cellular detachment and membrane degradation in neurons and the pathology-induced extracellular expression of latent and active MMPs. DIDS similarly inhibited extracellular MMP expression and cellular detachment induced by the pro-apoptotic agent staurosporine or the general proteinase agonist 4-aminophenylmercuric acetate (APMA). Conversely, DIDS-treatment did not impair stress- induced intracellular proMMP production, nor the intracellular cleavage of proMMP-2 to the active form, suggesting DIDS interferes with the vesicular extrusion of MMPs rather than directly inhibiting proteinase expression or activation. In support of this hypothesis, an antagonist of the V-type vesicular ATPase also inhibited extracellular MMP expression to a similar degree as DIDS. In addition, in a proteinase-independent model of vesicular exocytosis, DIDS prevented stimulus-evoked release of von Willebrand Factor from human umbilical vein endothelial cells. We conclude that DIDS inhibits MMP exocytosis and through this mechanism preserves neuronal membrane integrity during pathological stress.

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Buck LT, Hogg DW, Rodgers-Garlick C and Pamenter ME, (2012). Oxygen sensitive synaptic neurotransmission in anoxia-tolerant turtle cerebrocortex. Advances in Experimental Medicine and Biology. 758:71-79.

Abstract:

Anoxia rapidly elicits hyper-excitability and cell death in mammal brain but this is not so in anoxia-tolerant turtle brain where spontaneous electrical activity is suppressed by anoxia (i.e. spike arrest; SA). In anoxic turtle brain extracellular GABA concentrations increase dramatically and impact GABAergic synaptic transmission in a way that results in SA. Here we briefly review what is known about the regulation of glutamatergic signalling during anoxia and investigate the possibility that in anoxic turtle cortical neurons GABAA/B receptors play an important role in neuroprotection. Both AMPA and NMDA receptor currents decrease by about 50% in anoxic turtle cerebrocortex and therefore exhibit channel arrest, whereas GABA-A receptor currents increase twofold and increase whole-cell conductance. The increased post synaptic GABA-A receptor current is contrary to the channel arrest hypothesis but it does serve an important function. The reversal potential of the GABA-A receptor (EGABA) is only slightly depolarized relative to the resting membrane potential of the neuron and not sufficient to elicit an action potential. Therefore, when GABA-A receptors are activated, membrane potential moves to EGABA and prevents further depolarization by glutamatergic inputs during anoxia by a process termed shunting inhibition. Furthermore we discuss the presynaptic role of GABA-B receptors and show that increased endogenous GABA release during anoxia mediates SA by activating both GABA-A and B receptors and that this represents a natural oxygen-sensitive adaptive mechanism to protect brain from anoxic injury.

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Pamenter ME, Ali SS, Tang Q, Finley JC, Gu XQ, Dugan LL and Haddad GG (2012). An in vitro ischemic penumbral mimic perfusate increases NADPH oxidase-mediated superoxide production in cultured hippocampal neurons. Brain Research. 1452:165-172.

Abstract:

The currently accepted scheme for reactive oxygen species production during ischemia/ reperfusion injury is characterized by a deleterious mitochondria-derived burst of radical generation during reperfusion; however, recent examination of the penumbra suggests a central role for NADPH-oxidase (Nox)-mediated radical generation during the ischemic pe- riod. Therefore, we utilized a novel in vitro model of the penumbra to examine the free rad- ical profile of ischemic murine hippocampal neurons using electron paramagnetic resonance spectroscopy, and also the role of Nox in this generation and in cell fate. We re- port that free radical production increased ~75% at 2 h of ischemia, and this increase was abolished by: (1) scavenging of extracellular free radicals with superoxide dismutase (SOD), (2) a general anion channel antagonist, or (3) the Nox inhibitor apocynin. Similarly, at 24 h of ischemia, [ATP] decreased >95% and vital dye uptake increased 6-fold relative to controls; whereas apocynin, the Cl− channel antagonist 5-nitro-2-(3-phenylpropylamino)- benzoate (NPPB), or the free radical scavenger N-acetyl cysteine (NAC) each provided mod- erate neuroprotection, ameliorating 13–32% of [ATP]-depletion and 19–56% of vital dye up- take at 24 h. Our results support a cytotoxic role for Nox-mediated free radical production from penumbral neurons during the ischemic period.

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Mittal M, Gu XQ, Pak O, Pamenter ME, Haag D, Fuchs DB, Schermuly RT, Ghofrani HA, Brandes RP, Seeger W, Grimminger F, Haddad GG and Weissmann N (2012). Hypoxia induces Kv channel current inhibition by increased NAPDH oxidase-derived reactive oxygen species. Free Radicals in Biology and Medicine. 52(6):1033-1042.

Abstract:

There is current discussion whether reactive oxygen species are up- or downregulated in the pulmonary cir- culation during hypoxia, from which sources (i.e., mitochondria or NADPH oxidases) they are derived, and what the downstream targets of ROS are. We recently showed that the NADPH oxidase homolog NOX4 is upregulated in hypoxia-induced pulmonary hypertension in mice and contributes to the vascular remodeling in pulmonary hypertension. We here tested the hypothesis that NOX4 regulates Kv channels via an increased ROS formation after prolonged hypoxia. We showed that (1) NOX4 is upregulated in hypoxia-induced pul- monary hypertension in rats and isolated rat pulmonary arterial smooth muscle cells (PASMC) after 3 days of hypoxia, and (2) that NOX4 is a major contributor to increased reactive oxygen species (ROS) after hypox- ia. Our data indicate colocalization of Kv1.5 and NOX4 in isolated PASMC. The NADPH oxidase inhibitor and ROS scavenger apocynin as well as NOX4 siRNA reversed the hypoxia-induced decrease in Kv current density whereas the protein levels of the channels remain unaffected by siNOX4 treatment. Determination of cysteine oxidation revealed increased NOX4-mediated Kv1.5 channel oxidation. We conclude that sustained hypoxia de- creases Kv channel currents by a direct effect of a NOX4-derived increase in ROS.

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2011

Wilkie MP, Pamenter ME, Duquette S, Dhiyebi H, Sangha N, Skelton G, Smith MD and Buck LT (2011). The relationship between NMDA receptor function and the high ammonia tolerance of anoxia-tolerant goldfish (Carassius auratus Linnaeus). Journal of Experimental Biology. 214:4107-4120.

Summary:

Acute ammonia toxicity in vertebrates is thought to be characterized by a cascade of deleterious events resembling those associated with anoxic/ischemic injury in the central nervous system. A key event is the over-stimulation of neuronal N-methyl-D- aspartate (NMDA) receptors, which leads to excitotoxic cell death. The similarity between the responses to acute ammonia toxicity and anoxia suggests that anoxia-tolerant animals such as the goldfish (Carassius auratus Linnaeus) may also be ammonia tolerant. To test this hypothesis, the responses of goldfish were compared with those of the anoxia-sensitive rainbow trout (Oncorhynchus mykiss Walbaum) during exposure to high external ammonia (HEA). Acute toxicity tests revealed that goldfish are ammonia tolerant, with 96 h median lethal concentration (LC50) values of 199µmoll–1 and 4132µmoll–1 for NH3 and total ammonia ([TAmm]=[NH3]+[NH4+]), respectively. These values were ~5–6 times greater than corresponding NH3 and TAmm LC50 values measured in rainbow trout. Further, the goldfish readily coped with chronic exposure to NH4Cl (3–5µmoll–1) for 5 days, despite 6- fold increases in plasma [TAmm] to ~1300µmoll–1 and 3-fold increases in brain [TAmm] to 6700µmoll–1. Muscle [TAmm] increased by almost 8-fold from ~900µmolkg–1 wet mass (WM) to greater than 7000µmolkg–1 WM by 48h, and stabilized. Although urea excretion rates (JUrea) increased by 2–3-fold during HEA, the increases were insufficient to offset the inhibition of ammonia excretion that occurred, and increases in urea were not observed in the brain or muscle. There was a marked increase in brain glutamine concentration at HEA, from ~3000µmolkg–1 WM to 15,000µmolkg–1 WM after 48 h, which is consistent with the hypothesis that glutamine production is associated with ammonia detoxification. Injection of the NMDA receptor antagonists MK801 (0.5–8µmgkg–1) or ethanol (1–8µmgkg–1) increased trout survival time by 1.5–2.0-fold during exposure to 2µmoll–1 ammonia, suggesting that excitotoxic cell death contributes to ammonia toxicity in this species. In contrast, similar doses of MK801 or ethanol had no effect on ammonia-challenged (8–9.5µmoll–1 TAmm) goldfish survival times, suggesting that greater resistance to excitotoxic cell death contributes to the high ammonia-tolerance of the goldfish. Whole-cell recordings measured in isolated brain slices of goldfish telencephalon during in vitro exposure to 5µmoll–1 or 10µmoll–1 TAmm reversibly potentiated NMDA receptor currents. This observation suggested that goldfish neurons may not be completely resistant to ammonia-induced excitotoxicity. Subsequent western blot and densitometric analyses revealed that NMDA receptor NR1 subunit abundance was 40–60% lower in goldfish exposed to 3–5µmoll–1 TAmm for 5 days, which was followed by a restoration of NR1 subunit abundance after 3 days recovery in ammonia-free water. We conclude that the goldfish brain may be protected from excitotoxicity by downregulating the abundance of functional NMDA receptors during periods when it experiences increased internal ammonia.

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Pamenter ME, Hogg DW, Ormond J, Shin DS, Woodin MA and Buck LT (2011). Endogenous GABA(A) and GABA(B) receptor-mediated electrical suppression is critical to neuronal anoxia tolerance. Proceedings of the National Academy of Sciences (USA). 108(27):11274-11279.

Abstract:

Anoxic insults cause hyperexcitability and cell death in mammalian neurons. Conversely, in anoxia-tolerant turtle brain, spontaneous electrical activity is suppressed by anoxia (i.e., spike arrest; SA) and cell death does not occur. The mechanism(s) of SA is unknown but likely involves GABAergic synaptic transmission, because GABA concentration increases dramatically in anoxic turtle brain. We investigated this possibility in turtle cortical neurons exposed to anoxia and/or GABAA/B receptor (GABAR) modulators. Anoxia increased endogenous slow phasic GABAergic activity, and both anoxia and GABA reversibly induced SA by increasing GABAAR- mediated postsynaptic activity and Cl− conductance, which elimi- nated the Cl− driving force by depolarizing membrane potential (∼8 mV) to GABA receptor reversal potential (∼−81 mV), and dampened excitatory potentials via shunting inhibition. In addi- tion, both anoxia and GABA decreased excitatory postsynaptic activity, likely via GABABR-mediated inhibition of presynaptic glu- tamate release. In combination, these mechanisms increased the stimulation required to elicit an action potential >20-fold, and ex- citatory activity decreased >70% despite membrane potential de- polarization. In contrast, anoxic neurons cotreated with GABAA+BR antagonists underwent seizure-like events, deleterious Ca2+ influx, and cell death, a phenotype consistent with excitotoxic cell death in anoxic mammalian brain. We conclude that increased endoge- nous GABA release during anoxia mediates SA by activating an inhibitory postsynaptic shunt and inhibiting presynaptic gluta- mate release. This represents a natural adaptive mechanism in which to explore strategies to protect mammalian brain from low-oxygen insults.

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2008

Pamenter ME, and Buck LT (2008). delta-Opioid receptor antagonism induces NMDA receptor-dependent excitotoxicity in anoxic turtle cortex. Journal of Experimental Biology. 211:3512-3517.

Abstract:

δ-Opioid receptor (DOR) activation is neuroprotective against short-term anoxic insults in the mammalian brain. This protection may be conferred by inhibition of N-methyl-D-aspartate receptors (NMDARs), whose over-activation during anoxia otherwise leads to a deleterious accumulation of cytosolic calcium ([Ca2+]c), severe membrane potential (Em) depolarization and excitotoxic cell death (ECD). Conversely, NMDAR activity is decreased by ~50% with anoxia in the cortex of the painted turtle, and large elevations in [Ca2+]c, severe Em depolarization and ECD are avoided. DORs are expressed in high quantity throughout the turtle brain relative to the mammalian brain; however, the role of DORs in anoxic NMDAR regulation has not been investigated in turtles. We examined the effect of DOR blockade with naltrindole (1–10μmoll–1) on Em, NMDAR activity and [Ca2+]c homeostasis in turtle cortical neurons during normoxia and the transition to anoxia. Naltrindole potentiated normoxic NMDAR currents by 78±5% and increased [Ca2+]c by 13±4%. Anoxic neurons treated with naltrindole were strongly depolarized, NMDAR currents were potentiated by 70±15%, and [Ca2+]c increased 5-fold compared with anoxic controls. Following naltrindole washout, Em remained depolarized and [Ca2+]c became further elevated in all neurons. The naltrindole-mediated depolarization and increased [Ca2+]c were prevented by NMDAR antagonism or by perfusion of the Gi protein agonist mastoparan-7, which also reversed the naltrindole-mediated potentiation of NMDAR currents. Together, these data suggest that DORs mediate NMDAR activity in a Gi-dependent manner and prevent deleterious NMDAR-mediated [Ca2+]c influx during anoxic insults in the turtle cortex.

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Wilkie MP, Pamenter ME, Alkabie S, Carapic D, Shin DS and Buck LT (2008). Evidence of anoxia-induced channel arrest in the brain of the goldfish (Carassius auratus). Comparative Biochemistry and Physiology C. 148(4):355-362.

Abstract:

Acute ammonia toxicity in vertebrates is thought to be characterized by a cascade of deleterious events resembling those associated with anoxic/ischemic injury in the central nervous system. A key event is the over-stimulation of neuronal N-methyl-D- aspartate (NMDA) receptors, which leads to excitotoxic cell death. The similarity between the responses to acute ammonia toxicity and anoxia suggests that anoxia-tolerant animals such as the goldfish (Carassius auratus Linnaeus) may also be ammonia tolerant. To test this hypothesis, the responses of goldfish were compared with those of the anoxia-sensitive rainbow trout (Oncorhynchus mykiss Walbaum) during exposure to high external ammonia (HEA). Acute toxicity tests revealed that goldfish are ammonia tolerant, with 96 h median lethal concentration (LC50) values of 199µmoll–1 and 4132µmoll–1 for NH3 and total ammonia ([TAmm]=[NH3]+[NH4+]), respectively. These values were ~5–6 times greater than corresponding NH3 and TAmm LC50 values measured in rainbow trout. Further, the goldfish readily coped with chronic exposure to NH4Cl (3–5µmoll–1) for 5 days, despite 6- fold increases in plasma [TAmm] to ~1300µmoll–1 and 3-fold increases in brain [TAmm] to 6700µmoll–1. Muscle [TAmm] increased by almost 8-fold from ~900µmolkg–1 wet mass (WM) to greater than 7000µmolkg–1 WM by 48h, and stabilized. Although urea excretion rates (JUrea) increased by 2–3-fold during HEA, the increases were insufficient to offset the inhibition of ammonia excretion that occurred, and increases in urea were not observed in the brain or muscle. There was a marked increase in brain glutamine concentration at HEA, from ~3000µmolkg–1 WM to 15,000µmolkg–1 WM after 48 h, which is consistent with the hypothesis that glutamine production is associated with ammonia detoxification. Injection of the NMDA receptor antagonists MK801 (0.5–8µmgkg–1) or ethanol (1–8µmgkg–1) increased trout survival time by 1.5–2.0-fold during exposure to 2µmoll–1 ammonia, suggesting that excitotoxic cell death contributes to ammonia toxicity in this species. In contrast, similar doses of MK801 or ethanol had no effect on ammonia-challenged (8–9.5µmoll–1 TAmm) goldfish survival times, suggesting that greater resistance to excitotoxic cell death contributes to the high ammonia-tolerance of the goldfish. Whole-cell recordings measured in isolated brain slices of goldfish telencephalon during in vitro exposure to 5µmoll–1 or 10µmoll–1 TAmm reversibly potentiated NMDA receptor currents. This observation suggested that goldfish neurons may not be completely resistant to ammonia-induced excitotoxicity. Subsequent western blot and densitometric analyses revealed that NMDA receptor NR1 subunit abundance was 40–60% lower in goldfish exposed to 3–5µmoll–1 TAmm for 5 days, which was followed by a restoration of NR1 subunit abundance after 3 days recovery in ammonia-free water. We conclude that the goldfish brain may be protected from excitotoxicity by downregulating the abundance of functional NMDA receptors during periods when it experiences increased internal ammonia.

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Pamenter ME, and Buck LT (2008). Neuronal membrane potential is mildly depolarized in the anoxic turtle cortex. Comparative Biochemistry and Physiology A. 150(4):410-414.

Abstract:

Neuronal membrane potential (Em) regulates the activity of excitatory voltage-sensitive channels. Anoxic insults lead to a severe loss of Em and excitotoxic cell death (ECD) in mammalian neurons. Conversely, anoxia-tolerant freshwater turtle neurons depress energy usage during anoxia by altering ionic conductance to reduce neuronal excitability and ECD is avoided. This wholesale alteration of ion channel and pump activity likely has a significant effect on Em. Using the whole-cell patch clamp technique we recorded changes in Em from turtle cortical neurons during a normoxic to anoxic transition in the presence of various ion channel/pump modulators. Em did not change with normoxic perfusion but underwent a reversible, mild depolarization of 8.1±0.2 mV following anoxic perfusion. This mild anoxic depolarization (MAD) was not prevented by the manipulation of any single ionic conductance, but was partially reduced by pre-treatment with antagonists of GABAA receptors (5.7 ± 0.5 mV), cellular bicarbonate production (5.3 ± 0.2 mV) or K+ channels (6.0 ± 0.2 mV), or by perfusion of reactive oxygen species scavengers (5.2 ± 0.3 mV). Furthermore, all of these treatments induced depolarization in normoxic neurons. Together these data suggest that the MAD may be due to the summation of numerous altered ion conductance states during anoxia.

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Pamenter ME, Hogg DW and Buck LT (2008). Endogenous reductions in N-methyl-d-aspartate receptor activity inhibit nitric oxide production in the anoxic freshwater turtle cortex. FEBS Letters. 582(12):1738-1742.

Abstract:

Increased nitric oxide (NO) production from hypoxic mammalian neurons increases cerebral blood flow (CBF) but also glutamatergic excitotoxicity and DNA fragmentation. Anoxia- tolerant freshwater turtles have evolved NO-independent mecha- nisms to increase CBF; however, the mechanism(s) of NO regu- lation are not understood. In turtle cortex, anoxia or NMDAR blockade depressed NO production by 27 ± 3% and 41 ± 5%, respectively. NMDAR antagonists also reduced the subsequent anoxic decrease in NO by 74 ± 6%, suggesting the majority of the anoxic decrease is due to endogenous suppression of NMDAR activity. Prevention of NO-mediated damage during the transition to and from anoxia may be incidental to natural reductions of NMDAR activity in the anoxic turtle cortex.

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Pamenter ME, Shin DS and Buck LT (2008). Adenosine A1 receptor activation mediates NMDA receptor activity in a pertussis toxin-sensitive manner during normoxia but not anoxia in turtle cortical neurons. Brain Research. 1213:27-34.

Abstract:

Adenosine is a defensive metabolite that is critical to anoxic neuronal survival in the freshwater turtle. Channel arrest of the N-methyl-D-aspartate receptor (NMDAR) is a hallmark of the turtle’s remarkable anoxia tolerance and adenosine A1 receptor (A1R)-mediated depression of normoxic NMDAR activity is well documented. However, experiments examining the role of A1Rs in regulating NMDAR activity during anoxia have yielded inconsistent results. The aim of this study was to examine the role of A1Rs in the normoxic and anoxic regulation of turtle brain NMDAR activity. Whole-cell NMDAR currents were recorded for up to 2 h from turtle cortical pyramidal neurons exposed to pharmacological A1R or Gi protein modulation during normoxia (95% O2/5% CO2) and anoxia (95% N2/5% CO2). NMDAR currents were unchanged during normoxia and decreased 51 ± 4% following anoxic exposure. Normoxic agonism of A1Rs with adenosine or N6-cyclopentyladenosine (CPA) decreased NMDAR currents 57±11% and 59±6%, respectively. The A1R antagonist 8-cyclopentyl-1,3-dimethylxanthine (DPCPX) had no effect on normoxic NMDAR currents and prevented the adenosine and CPA-mediated decreases in NMDAR activity. DPCPX partially reduced the anoxic decrease at 20 but not 40 min of treatment. The Gi protein inhibitor pertussis toxin (PTX) prevented both the CPA and anoxia-mediated decreases in NMDAR currents and calcium chelation or blockade of mitochondrial ATP-sensitive K+ channels also prevented the CPA-mediated decreases. Our results suggest that the long-term anoxic decrease in NMDAR activity is activated by a PTX-sensitive mechanism that is independent of A1R activity.

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Pamenter ME, Shin DS, Cooray M and Buck LT (2008). Mitochondrial ATP-sensitive K+ channels regulate NMDAR activity in the cortex of the anoxic western painted turtle. Journal of Physiology (London). 586(4):1043-1058.

Abstract:

Hypoxic mammalian neurons undergo excitotoxic cell death, whereas painted turtle neurons survive prolonged anoxia without apparent injury. Anoxic survival is possibly mediated by a decrease in N-methyl-D-aspartate receptor (NMDAR) activity and maintenance of cellular calcium concentrations ([Ca2+]c) within a narrow range during anoxia. In mammalian ischaemic models, activation of mitochondrial ATP-sensitive K+ (mKATP) channels partially uncouples mitochondria resulting in a moderate increase in [Ca2+]c and neuro-protection. The aim of this study was to determine the role of mKATP channels in anoxic turtle NMDAR regulation and if mitochondrial uncoupling and [Ca2+]c changes underlie this regulation. In isolated mitochondria, the KATP channel activators diazoxide and levcromakalim increased mitochondrial respiration and decreased ATP production rates, indicating mitochondria were ‘mildly’ uncoupled by 10–20%. These changes were blocked by the mKATP antagonist 5-hydroxydecanoic acid (5HD). During anoxia, [Ca2 + ]c increased 9.3±0.3% and NMDAR currents decreased 48.9±4.1%. These changes were abolished by K channel blockade with 5HD or glibenclamide, Ca2+ chelation with ATP c 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) or by activation of the mitochondrial Ca2+ uniporter with spermine. Similar to anoxia, diazoxide or levcromakalim increased [Ca2+]c 8.9 ± 0.7% and 3.8 ± 0.3%, while decreasing normoxic whole-cell NMDAR currents by 41.1 ± 6.7% and 55.4 ± 10.2%, respectively. These changes were also blocked by 5HD or glibenclamide, BAPTA, or spermine. Blockade of mitochondrial Ca2+-uptake decreased normoxic NMDAR currents 47.0 ± 3.1% and this change was blocked by BAPTA but not by 5HD. Taken together, these data suggest mKATP channel activation in the anoxic turtle cortex uncouples mitochondria and reduces mitochondrial Ca2+ uptake via the uniporter, subsequently increasing [Ca2+]c and decreasing NMDAR activity.

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Pamenter ME, Shin DS and Buck LT (2008). AMPA receptors undergo channel arrest in the anoxic turtle cortex. American Journal of Physiology: Regulatory, Integrative and Comparative Physiology. 294(2):R606-R613.

Abstract:

AMP A receptors undergo channel arrest in the anoxic turtle cortex. Am J Physiol Regul Integr Comp Physiol 294: R606–R613, 2008. First published December 5, 2007; doi:10.1152/ajpregu.00433.2007.—Without ox- ygen, all mammals suffer neuronal injury and excitotoxic cell death mediated by overactivation of the glutamatergic N-methyl-D-aspartate receptor (NMDAR). The western painted turtle can survive anoxia for months, and downregulation of NMDAR activity is thought to be neuroprotective during anoxia. NMDAR activity is related to the activity of another glutamate receptor, the α-amino-3-hydroxy-5- methylisoxazole-4-propionic acid receptor (AMPAR). AMPAR blockade is neuroprotective against anoxic insult in mammals, but the role of AMPARs in the turtle’s anoxia tolerance has not been inves- tigated. To determine whether AMPAR activity changes during hyp- oxia or anoxia in the turtle cortex, whole cell AMPAR currents, AMPAR-mediated excitatory postsynaptic potentials (EPSPs), and excitatory postsynaptic currents (EPSCs) were measured. The effect of AMPAR blockade on normoxic and anoxic NMDAR currents was also examined. During 60 min of normoxia, evoked peak AMPAR currents and the frequencies and amplitudes of EPSPs and EPSCs did not change. During anoxic perfusion, evoked AMPAR peak currents decreased 59.2 ± 5.5 and 60.2 ± 3.5% at 20 and 40 min, respectively. EPSP frequency (EPSPƒ) and amplitude decreased 28.7 ± 6.4% and 13.2 ± 1.7%, respectively, and EPSCƒ and amplitude decreased 50.7 ± 5.1% and 51.3 ± 4.7%, respectively. In contrast, hypoxic (PO2 ± 5%) AMPAR peak currents were potentiated 56.6 ± 20.5 and 54.6 ± 15.8% at 20 and 40 min, respectively. All changes were reversed by reoxygenation. AMPAR currents and EPSPs were abol- ished by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). In neurons pretreated with CNQX, anoxic NMDAR currents were reversibly depressed by 49.8 ± 7.9%. These data suggest that AMPARs may undergo channel arrest in the anoxic turtle cortex.

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2007

Pamenter ME, Richards MD and Buck LT (2007). Anoxia-induced changes in reactive oxygen species and cyclic nucleotides in the painted turtle. Journal of Comparative Physiology B. 177(4):473-481.

Abstract:

The Western painted turtle survives months without oxygen. A key adaptation is a coordinated reduc- tion of cellular ATP production and utilization that may be signaled by changes in the concentrations of reactive oxy- gen species (ROS) and cyclic nucleotides (cAMP and cGMP). Little is known about the involvement of cyclic nucleotides in the turtle’s metabolic arrest and ROS have not been previously measured in any facultative anaerobes. The present study was designed to measure changes in these second messengers in the anoxic turtle. ROS were measured in isolated turtle brain sheets during a 40-min normoxic to anoxic transition. Changes in cAMP and cGMP were determined in turtle brain, pectoralis muscle, heart and liver throughout 4 h of forced submergence at 20–22°C. Turtle brain ROS production decreased 25% within 10min of cyanide or N2-induced anoxia and returned to control levels upon reoxygenation. Inhibition of electron transfer from ubiquinol to complex III caused a smaller decrease in [ROS]. Conversely, inhibition of com- plex I increased [ROS] 15% above controls. In brain [cAMP] decreased 63%. In liver [cAMP] doubled after 2 h of anoxia before returning to control levels with prolonged anoxia. Conversely, skeletal muscle and heart [cAMP] remained unchanged; however, skeletal muscle [cGMP] became elevated sixfold after 4 h of submergence. In liver and heart [cGMP] rose 41 and 127%, respectively, after 2 h of anoxia. Brain [cGMP] did not change significantly during 4 h of submergence. We conclude that turtle brain ROS production occurs primarily between mitochondrial complexes I and III and decreases during anoxia. Also, cyclic nucleotide concentrations change in a manner sug- gestive of a role in metabolic suppression in the brain and a role in increasing liver glycogenolysis.

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2006

Walsh PJ, Veauvy CM, McDonald MD, Pamenter ME, Buck LT and Wilkie MP (2006). Piscine insights into comparisons of anoxia tolerance, ammonia toxicity, stroke and hepatic encephalopathy. Comparative Biochemistry and Physiology A. 147(2):332-343.

Abstract:

Although the number of fish species that have been studied for both hypoxia/anoxia tolerance and ammonia tolerance are few, there appears to be a correlation between the ability to survive these two insults. After establishing this correlation with examples from the literature, and after examining the role Peter Lutz played in catalyzing this convergent interest in two variables, this article explores potential mechanisms underpinning this correlation. We draw especially on the larger body of information for two human diseases with the same effected organ (brain), namely stroke and hepatic encephalopathy. While several dissimilarities exist between the responses of vertebrates to anoxia and hyperammonemia, one consistent observation in both conditions is an overactivation of NMDA receptors or glutamate neurotoxicity. We propose a glutamate excitotoxicity hypothesis to explain the correlation between ammonia and hypoxia resistance in fish. Furthermore, we suggest several experimental paths to test this hypothesis.

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Buck LT and Pamenter ME, (2006). Adaptive responses of vertebrate neurons to anoxia – matching supply to demand. Respiratory Physiology and Neurobiology. 154(1-2):226-240.

Abstract:

Oxygen depleted environments are relatively common on earth and represent both a challenge and an opportunity to organisms that survive there. A commonly observed survival strategy to this kind of stress is a lowering of metabolic rate or metabolic depression. Whether metabolic rate is at a normal or a depressed level the supply of ATP (glycolysis and oxidative phosphory- lation) must match the cellular demand for ATP (protein synthesis and ion pumping), a condition that must of course be met for long-term survival in hypoxic and anoxic environments. Underlying a decrease in metabolic rate is a corresponding decrease in both ATP supply and ATP demand pathways setting a new lower level for ATP turnover. Both sides of this equation can be actively regulated by second messenger pathways but it is less clear if they are regulated differentially or even sequentially with the onset of anoxia. The vertebrate brain is extremely sensitive to low oxygen levels yet some species can survive in oxygen depleted environments for extended periods and offer a working model of brain survival without oxygen. Hypoxia tolerant vertebrate brain will be the primary focus of this review; however, we will draw upon research involving hypoxia/ischemia tolerance mechanisms in liver and heart to offer clues to how brain can tolerate anoxia. The issue of regulating ATP supply or demand pathways will also be addressed with a focus on ion channel arrest being a significant mechanism to reduce ATP demand and therefore metabolic rate. Furthermore, mitochondria are ideally situated to serve as cellular oxygen sensors and mediator of protective mechanisms such as ion channel arrest. Therefore, we will also describe a mitochondria based mechanism of ion channel arrest involving ATP-sensitive mitochondrial K+ channels, cytosolic calcium and reaction oxygen species concentrations.

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2005

Shin DS, Wilkie MP, Pamenter ME, and Buck LT (2005). Calcium and protein phosphatase 1/2A attenuate N-methyl-D-aspartate receptor activity in the anoxic turtle cortex. Comparative Biochemistry and Physiology A. 142(1):50-57.

Abstract:

Excitotoxic cell death (ECD) is characteristic of mammalian brain following min of anoxia, but is not observed in the western painted turtle following days to months without oxygen. A key event in ECD is a massive increase in intracellular Ca2+ by over-stimulation of N- methyl-d-aspartate receptors (NMDARs). The turtle’s anoxia tolerance may involve the prevention of ECD by attenuating NMDAR-induced Ca2+ influx. The goal of this study was to determine if protein phosphatases (PPs) and intracellular calcium mediate reductions in turtle cortical neuron whole-cell NMDAR currents during anoxia, thereby preventing ECD. Whole-cell NMDAR currents did not change during 80 min of normoxia, but decreased 56% during 40 min of anoxia. Okadaic acid and calyculin A, inhibitors of serine/threonine PP1 and PP2A, potentiated NMDAR currents during normoxia and prevented anoxia-mediated attenuation of NMDAR currents. Decreases in NMDAR activity during anoxia were also abolished by inclusion of the Ca2+ chelator — BAPTA and the calmodulin inhibitor — calmidazolium. However, cypermethrin, an inhibitor of the Ca2+/calmodulin-dependent PP2B (calcineurin), abolished the anoxic decrease in NMDAR activity at 20, but not 40 min suggesting that this phosphatase might play an early role in attenuating NMDAR activity during anoxia. Our results show that PPs, Ca2+ and calmodulin play an important role in decreasing NMDAR activity during anoxia in the turtle cortex. We offer a novel mechanism describing this attenuation in which PP1 and 2A dephosphorylate the NMDAR (NR1 subunit) followed by calmodulin binding, a subsequent dissociation of a-actinin-2 from the NR1 subunit, and a decrease in NMDAR activity.

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Book Chapters

paper iconPamenter ME (2016). Delta-opioid receptors and glutamatergic toxicity. Neural function of the delta-opioid receptor. New York, USA: Springer Publishing (Y. Xia, editor).

paper icon Pamenter ME and Baldy C (2017). Chronic intermittent hypoxia contributes to sleep-disordered breathing in infants and adults. Berlin, GER: Avid Science (S. Ravini, editor).
paper icon Borecky LG and Pamenter ME (2017). Oxygen sensing and transcriptional regulation of adaptive responses to hypoxia. New York, USA: Nova Science (MJ. Cosgrove, editor).
paper icon Pamenter ME (2015). High-throughput cell death assays. Neuronal Cell Death – Methods and Protocols. New York, USA: Springer Publishing (J. Walker, editor).
paper icon Pamenter ME and Ali SS (2014). Registering superoxide production in live neuronal culture by EPR. Free Radicals: The Role of Antioxidants and Pro-oxidants in Cancer Development. New York, USA: Nova Science (W Stone, editor).
paper icon Buck LT, Hogg DW, Rodgers C and Pamenter ME (2012). The Painted Turtle as a model of natural anoxia tolerance: role of the neurotransmitter GABA. Turtles: Anatomy, Ecology and Conservation. New York, USA: Nova Science (MJ Cosgrove, SA Roe, editors).
paper icon Buck LT, Hogg DW, Rodgers-Garlick C and Pamenter ME (2011). Oxygen sensitive synaptic neurotransmission in anoxia-tolerant turtle cerebrocortex. Arterial Chemoreception: From Molecules to Systems. New York, USA: Springer Publishing (N. Gotsiridze-Columbus, editor)