Leslie Thomas Buck

Effects of lsoflurane and Hypothermia on Glutamate Receptor-mediated Calcium Influx in Brain Slices

Background:To understand how volatile anesthetics protect neurons during cerebral ischemia, we studied the effects of isoflurane on cerebral glutamate receptor-mediated calcium influx. Calcium influx via these key excitatory receptors may mediate pain transmission, memory, and the pathophyslologic sequelae of cerebral anoxia or ischemia. Because cerebral protection by hypothermia may involve a decrease in glutamate receptor activity, we also examined the interaction of temperature and isoflurane on glutamate receptor inhibition. Methods:We measured glutamate receptor-mediated changes in cytosolic calcium in 300-µm-thlck rat cortical brain slices. Temperature was varied to 28, 34, 37, or 39°C and isoflurane partial pressure to 0.016-0.019 atm (equivalent to 1.16 minimum alveolar concentration [MAC], adjusted for temperature and age). Brain slices were loaded with fura-2 to permit measurement of cytosolic free calcium. Calcium changes due to the glutamate receptor agonist N-methyl-D-aspartate (NMDA) (50 µM), to ischemia levels of L-glutamate (1.0 mM) or to simulated ischemia (1.0 mM glutamate, 100 µM NaCN, and 3.5 mM iodoacetate) was then measured. Slice lactate dehydrogenase leakage and adenosine triphosphate were measured as indices of cellular integrity. Results: Isoflurane reduced both L-glutamate and NMDA-mediated calcium fluxes by approximately 60%. Neither the activity of the NMDA receptor nor its inhibition by isoflurane was altered by temperature. The rate of calcium influx during ischemia was significantly reduced both by temperature and by isoflurane (P < 0.05). Adenosine triphosphate loss and lactate dehydrogenase leakage were reduced by isoflurane during simulated ischemia by 37% and 73% (P < 0.05), respectively. Conclusions:(1) At 1.16 MAC, isoflurane potently Inhibits glutamate receptors and delays cellular injury induced by simulated ischemia, and (2) hypothermia does not reduce the Intrinsic activity of cortical glutamate receptors but delays calcium accumulation during simulated ischemia. Isoflurane reduces the severity of key pathophyslologic events in an in vitro model of simulated cerebral ischemia.

Volatile and Intravenous Anesthetics Decrease Glutamate Release from Cortical Brain Slices during Anoxia

Background Extracellular accumulation of the excitatory neurotransmitter L‐glutamate during cerebral hypoxia or ischemia contributes to neuronal death. Anesthetics inhibit release of synaptic neurotransmitters but it is unknown if they alter net extrasynaptic glutamate release, which accounts for most of the glutamate released during hypoxia or ischemia. The purpose of this study was to determine if different types of anesthetics decrease hypoxia‐induced glutamate release from rat brain slices.

Time-dependent expression of heat shock proteins 70 and 90 in tissues of the anoxic western painted turtle

SUMMARY Expression of the constitutive Hsp73, inducible Hsp72 and Hsp90 was investigated in brain, heart, liver and skeletal muscle of the anoxia-tolerant western painted turtle Chrysemys picta bellii in response to 2, 6, 12, 18, 24 and 30 h forced dives and following 1 h recovery from 12, 24 and 30 h forced dives at 17°C. During a dive, expression of all three Hsps examined remained at control levels for at least 12 h in all tissues examined except the liver, where Hsp72 showed a decrease at 12 h, reaching a significant threefold decrease by 24 h. Brain and liver Hsp73, 72 and 90 expression increased two- to threefold at 18, 24 and 30 h. Heart and muscle Hsp73 and heart Hsp90 expression remained at normoxic levels throughout the entire dive, while heart and muscle Hsp72 and muscle Hsp90 increased two- to fourfold at 24 and 30 h. Following reoxygenation, Hsp expression increased in all tissues examined. These data indicate that increased Hsp expression is not critical in the early adaptation to anoxic survival and that short-term anoxia is probably not a stress for species adapted to survive long periods without oxygen. However, the late upregulation of heat shock proteins during anoxia suggests that stress proteins play a role in promoting long-term anoxia tolerance.

Effect of Anoxia and Pharmacological Anoxia on Whole‐Cell NMDA Receptor Currents in Cortical Neurons from the Western Painted Turtle

The mammalian brain undergoes rapid cell death during anoxia that is characterized by uncontrolled Ca2+ entry via N‐methyl‐D‐aspartate receptors (NMDARs). In contrast, the western painted turtle is extremely anoxia tolerant and maintains close‐to‐normal [Ca2+]i during periods of anoxia lasting from days to months. A plausible mechanism of anoxic survival in turtle neurons is the regulation of NMDARs to prevent excitotoxic Ca2+ injury. However, studies using metabolic inhibitors such as cyanide (NaCN) as a convenient method to induce anoxia may not represent a true anoxic stress. This study was undertaken to determine whether turtle cortical neuron whole‐cell NMDAR currents respond similarly to true anoxia with N2 and to NaCN‐induced anoxia. Whole‐cell NMDAR currents were measured during a control N2‐induced anoxic transition and a control NaCN‐induced transition. During anoxia with N2 normalized, NMDAR currents decreased to 35.3% ± 10.8% of control values. Two different NMDAR current responses were observed during NaCN‐induced anoxia: one resulted in a 172% ± 51% increase in NMDAR currents, and the other was a decrease to 48% ± 14% of control. When responses were correlated to the two major neuronal subtypes under study, we found that stellate neurons responded to NaCN treatment with a decrease in NMDAR current, while pyramidal neurons exhibited both increases and decreases. Our results show that whole‐cell NMDAR currents respond differently to NaCN‐induced anoxia than to the more physiologically relevant anoxia with N2.

Endogenous GABA(A) and GABA(B) receptor-mediated electrical suppression is critical to neuronal anoxia tolerance.

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 eliminated 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 addition, both anoxia and GABA decreased excitatory postsynaptic activity, likely via GABABR-mediated inhibition of presynaptic glutamate release. In combination, these mechanisms increased the stimulation required to elicit an action potential >20-fold, and excitatory activity decreased >70% despite membrane potential depolarization. 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 endogenous GABA release during anoxia mediates SA by activating an inhibitory postsynaptic shunt and inhibiting presynaptic glutamate release. This represents a natural adaptive mechanism in which to explore strategies to protect mammalian brain from low-oxygen insults.

Molecular Adaptations for Survival during Anoxia: Lessons from Lower Vertebrates

Anoxia-tolerant neurons from several species of animals may offer unparalleled opportunities to identify strategies that might be employed to enhance the hypoxia or ischemia tolerance of vulnerable neurons. In this review, the authors describe how the response of hypoxia-tolerant neurons to limited oxygen supply involves a suite of mechanisms that reduce energy expenditure in concert with decreased energy availability. This response avoids energy depletion, excitotoxic neuronal death, and apoptosis. Suppression of ion channel functions, particularly those of the ionotropic glutamate receptors, is a response common in hypoxia-tolerant neurons. The depression of excitability thereby achieved is essential given that the fundamental response to oxygen lack in anoxia-tolerant cells is a throttling down of metabolism to “pilot-light” levels. Many different types of processes have been found to down-regulate ion channel function. These include phosphorylation control, interactions with intracellular and extracellular ions, removal of active receptors from the neurolemma, and the direct sensing of oxygen by Na+ and K+ channels. Changes in [Ca2+]i may initiate a protective down-regulation of many different pumps or channels. Transcriptional events leading to differential and/or decreased expression of receptors, proteins, and their subunits are probably very important but little studied.

Mitochondrial ATP-sensitive K+ channels regulate NMDAR activity in the cortex of the anoxic western painted turtle.

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 neuroprotection. 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 KATP channel blockade with 5HD or glibenclamide, Ca2+c chelation with 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.

delta-Opioid receptor antagonism induces NMDA receptor-dependent excitotoxicity in anoxic turtle cortex.

SUMMARY 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 (1–10 μmol l–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.

REVERSIBLE DECREASES IN ATP AND PCR CONCENTRATIONS IN ANOXIC TURTLE BRAIN

A hallmark of anoxia tolerance in western painted turtles is relative constancy of tissue adenylate concentrations during periods of oxygen limitation. During anoxia heart and brain intracellular compartments become more acidic and cellular energy demands are met by anaerobic glycolysis. Because changes in adenylates and pH during anoxic stress could represent important signals triggering metabolic and ion channel down-regulation we measured PCr, ATP and intracellular pH in turtle brain sheets throughout a 3-h anoxic-re-oxygenation transition with 31P NMR. Within 30 min of anoxia, PCr levels decrease 40% and remain at this level during anoxia. A different profile is observed for ATP, with a statistically significant decrease of 23% occurring gradually during 110 min of anoxic perfusion. Intracellular pH decreases significantly with the onset of anoxia, from 7.2 to 6.6 within 50 min. Upon re-oxygenation PCr, ATP and intracellular pH recover to pre-anoxic levels within 60 min. This is the first demonstration of a sustained reversible decrease in ATP levels with anoxia in turtle brain. The observed changes in pH and adenylates, and a probable concomitant increase in adenosine, may represent important metabolic signals during anoxia.

Excitatory actions of GABA mediate severe-hypoxia-induced depression of neuronal activity in the pond snail (Lymnaea stagnalis).

SUMMARY To characterize the effect of severe hypoxia on neuronal activity, long-term intracellular recordings were made from neurones in the isolated central ring ganglia of Lymnaea stagnalis. When a neurone at rest in normoxia was subjected to severe hypoxia, action potential firing frequency decreased by 38% (from 2.4-1.5 spikes s-1), and the resting membrane potential hyperpolarized from -70.3 to -75.1 mV. Blocking GABAA receptor-mediated synaptic transmission with the antagonist bicuculline methiodide (100 μmol l-1) decreased neuronal activity by 36%, and prevented any further changes in response to severe hypoxia, indicating that GABAergic neurotransmission mediates the severe hypoxia-induced decrease in neuronal activity. Puffing 100 μmol l-1 GABA onto the cell body produced an excitatory response characterized by a transient increase in action potential (AP) firing, which was significantly decreased in severe hypoxia. Perturbing intracellular chloride concentrations with the Na+/K+/Cl- (NKCC1) cotransporter antagonist bumetanide (100 μmol l-1) decreased AP firing by 40%, consistent with GABA being an excitatory neurotransmitter in the adult Lymnaea CNS. Taken together, these studies indicate that severe hypoxia reduces the activity of NKCC1, leading to a reduction in excitatory GABAergic transmission, which results in a hyperpolarization of the resting membrane potential (Vm) and as a result decreased AP frequency.