Environmental remodelling of GABAergic and glutamatergic neurotransmission: rise of the anoxia-tolerant turtle brain.

Climate cooling over the past one hundred thousand years has resulted in seasonal ice cover at northern and southern latitudes that has selected for hypoxia and anoxia tolerance in some species, such as freshwater turtles. At the northern reaches of their range, North American freshwater turtles spend 4 months or more buried in the mud bottom of ice covered lakes and ponds. From a comparative perspective this gives us the opportunity to understand how an extremely oxygen-sensitive organ, such as the vertebrate brain, can function without oxygen for long periods. Brain function is based on complex excitatory (on) and inhibitory (off) circuits involving the major neurotransmitters glutamate and, γ-aminobutyric acid (GABA) respectively. When a mammalian brain becomes anoxic, glutamate levels rise within minutes resulting in excitotoxic cell death which does not occur in anoxic turtle brain. The response in turtle brain has been remodelled - GABA levels rise rapidly resulting in large inhibitory GABA receptor currents and inhibition of glutamate receptor function that together depress neuronal activity.

Factors influencing cranial variation between prehistoric Japanese forager populations.

Understanding the factors shaping human crania has long been a goal of biological anthropology, and climate, diet, and population history are three of the most well-established influences. The effects of these factors are, however, rarely compared within a single, variable population, limiting interpretations of their relative contribution to craniofacial form. Jomon prehistoric foragers inhabited Japan throughout its climatic and ecological range and developed correspondingly varied modes of subsistence. We have previously demonstrated that a large sample of Jomon crania showed no clear climatic pattern; here, we examine variation in Jomon crania in more detail to determine if dietary factors and/or population history influence human intrapopulation variation at this scale. Based on well-established archaeological differences, we divide the Jomon into dietary groups and use geometric morphometric methods to analyse relationships between cranial shape, diet, and population history. We find evidence for diet-related influences on the shape of the neurocranium, particularly in the temporalis region. These shape differences may be interpreted in the context of regional variation in the biomechanical requirements of different diets. More experimental biomechanical and nutritional evidence is needed, however, to move suggested links between dietary content and cranial shape from plausible to well-supported. In contrast with the global scale of human variation, where neutral processes are the strongest influence on cranial shape, we find no pattern of population history amongst individuals from these Jomon sites. The determinants of cranial morphology are complex and the effect of diet is likely mediated by factors including sex, social factors, and chronology. Our results underline the subtlety of the effects of dietary variation beyond the forager/farmer dichotomy on cranial morphology and contribute to our understanding of the complexity of selective pressures shaping human phenotypes on different geographic scales. Supplementary Information: The online version contains supplementary material available at 10.1007/s12520-023-01901-6.

Transcriptome analysis of the central nervous system of the mollusc Lymnaea stagnalis.

BACKGROUND: The freshwater snail Lymnaea stagnalis (L. stagnalis) has served as a successful model for studies in the field of Neuroscience. However, a serious drawback in the molecular analysis of the nervous system of L. stagnalis has been the lack of large-scale genomic or neuronal transcriptome information, thereby limiting the use of this unique model. RESULTS: In this study, we report 7,712 distinct EST sequences (median length: 847 nucleotides) of a normalized L. stagnalis central nervous system (CNS) cDNA library, resulting in the largest collection of L. stagnalis neuronal transcriptome data currently available. Approximately 42% of the cDNAs can be translated into more than 100 consecutive amino acids, indicating the high quality of the library. The annotated sequences contribute 12% of the predicted transcriptome size of 20,000. Surprisingly, approximately 37% of the L. stagnalis sequences only have a tBLASTx hit in the EST library of another snail species Aplysia californica (A. californica) even using a low stringency e-value cutoff at 0.01. Using the same cutoff, approximately 67% of the cDNAs have a BLAST hit in the NCBI non-redundant protein and nucleotide sequence databases (nr and nt), suggesting that one third of the sequences may be unique to L. stagnalis. Finally, using the same cutoff (0.01), more than half of the cDNA sequences (54%) do not have a hit in nematode, fruitfly or human genome data, suggesting that the L. stagnalis transcriptome is significantly different from these species as well. The cDNA sequences are enriched in the following gene ontology functional categories: protein binding, hydrolase, transferase, and catalytic enzymes. CONCLUSION: This study provides novel molecular insights into the transcriptome of an important molluscan model organism. Our findings will contribute to functional analyses in neurobiology, and comparative evolutionary biology. The L. stagnalis CNS EST database is available at http://www.Lymnaea.org/.

Evidence of different climatic adaptation strategies in humans and non-human primates.

To understand human evolution it is critical to clarify which adaptations enabled our colonisation of novel ecological niches. For any species climate is a fundamental source of environmental stress during range expansion. Mammalian climatic adaptations include changes in size and shape reflected in skeletal dimensions and humans fit general primate ecogeographic patterns. It remains unclear however, whether there are also comparable amounts of adaptation in humans, which has implications for understanding the relative importance of biological/behavioural mechanisms in human evolution. We compare cranial variation between prehistoric human populations from throughout Japan and ecologically comparable groups of macaques. We compare amounts of intraspecific variation and covariation between cranial shape and ecological variables. Given equal rates and sufficient time for adaptation for both groups, human conservation of non-human primate adaptation should result in comparable variation and patterns of covariation in both species. In fact, we find similar amounts of intraspecific variation in both species, but no covariation between shape and climate in humans, contrasting with strong covariation in macaques. The lack of covariation in humans may suggest a disconnect in climatic adaptation strategies from other primates. We suggest this is due to the importance of human behavioural adaptations, which act as a buffer from climatic stress and were likely key to our evolutionary success.

50 years of comparative biochemistry: The legacy of Peter Hochachka.

Peter Hochachka was an early pioneer in the field of comparative biochemistry. He passed away in 2002 after 4 decades of research in the discipline. To celebrate his contributions and to coincide with what would have been his 80th birthday, a group of his former students organized a symposium that ran as a satellite to the 2017 Canadian Society of Zoologists annual meeting in Winnipeg, Manitoba (Canada). This Special Issue of CBP brings together manuscripts from symposium attendees and other authors who recognize the role Peter played in the evolution of the discipline. In this article, the symposium organizers and guest editors look back on his career, celebrating his many contributions to research, acknowledging his role in training of generations of graduate students and post-doctoral fellows in comparative biochemistry and physiology.

Adaptive responses of vertebrate neurons to anoxia--matching supply to demand.

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 phosphorylation) 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.

Hypoxia-induced silencing of NMDA receptors in turtle neurons.

Hypoxia-induced suppression of NMDA receptors (NMDARs) in western painted turtle (Chrysemys picta) cortical neurons may be critical for surviving months of anoxic dormancy. We report that NMDARs are silenced by at least three different mechanisms operating at different times during anoxia. In pyramidal neurons from cerebrocortex, 1-8 min anoxia suppressed NMDAR activity (Ca(2+) influx and open probability) by 50-60%. This rapid decrease in receptor activity was controlled by activation of phosphatase 1 or 2A but was not associated with an increase in [Ca(2+)](i). However, during 2 hr of anoxia, [Ca(2+)](i) in cerebrocortical neurons increased by 35%, and suppression of NMDARs was predicted by the increase of [Ca(2+)](i) and controlled by calmodulin. An additional mechanism of NMDAR silencing, reversible removal of receptors from the cell membrane, was found in cerebrocortex of turtles remaining anoxic at 3 degrees C for 3-21 d. When suppression of NMDARs was prevented with phosphatase inhibitors, tolerance of anoxia was lost. Silencing of NMDARs is thus critical to the remarkable ability of C. picta to tolerate life without oxygen.

Interactive effects of pH and temperature on N-methyl-D-aspartate receptor activity in rat cortical brain slices.

Low extracellular pH decreases the activity of the N-methyl-D-aspartate (NMDA) glutamate receptor, and may thus limit neuronal calcium overload during cerebral ischemia. During induced hypothermia, alkaline pH ("alphastat regulation") is often used to preserve cardiac and enzymatic function. The purpose of this study is to measure the functional activity of cerebral cortex NMDA receptors over the range of temperatures used in profound hypothermic cardiopulmonary bypass (20-37 degrees C). Extracellular pH was varied over a broad range relevant to both alphastat and pH stat acid-base management (7.0-7.8). Change in cytosolic free calcium evoked by 50 microM NMDA in brain slices was used as an index of NMDA receptor activity. Cortical slices (300 microns thick) were loaded with fura-2 Aspartate Methyl for study in a fluorometer. At 37 degrees C, a change in extracellular pH from 7.1 to 7.8 increased the NMDA-evoked change in cytosolic calcium in brain slices by a factor of 4 (p < 0.05). In contrast, at 20 degrees C there was minimal effect of changing extracellular pH from 7.1 to 7.8 (27% increase). We conclude that hypothermia results in decreased pH sensitivity of the NMDA receptor. The results predict that different strategies of pH management during induced hypothermia may have limited impact on NMDA receptor-mediated processes, such as neuronal calcium overload.

Succinate and alanine as anaerobic end-products in the diving turtle (Chrysemys picta bellii).

The western painted turtle is an extremely anoxia-tolerant vertebrate capable of tolerating blood lactate levels of 150-200 mM. Since lactate increases to such high levels, other fermentation end-products such as succinate and alanine, which have not been previously measured in this species, might also be expected to increase. Therefore, I measured turtle heart, liver, and blood concentrations of lactate, succinate, and alanine following a 28-day anoxic dive at 5 degrees C. Succinate and lactate concentrations increased significantly in all three compartments while alanine increased significantly in the liver only. Lactate was found to accumulate by a similar amount in all three compartments (66.4-80.5 micromol g or ml(-1) in the blood compartment) and was used as a reference to which alanine and succinate concentrations could be compared. Succinate and alanine levels increased by 2 and 0.9% of lactate in liver, approximately 0.3 and 0.04% of lactate in blood, and 0.6 and 0.07% of lactate in heart, respectively. The contribution of each to the total anoxic heat production was calculated and accounted for an additional 1.5% of the previously measured exothermic gap. I conclude that succinate and alanine concentrations do increase in the anoxic turtle but are minor anaerobic end-products.

Oxygen-sensitive reduction in Ca²âº-activated K⁺ channel open probability in turtle cerebrocortex.

In response to low ambient oxygen levels the western painted turtle brain undergoes a large depression in metabolic rate which includes a decrease in neuronal action potential frequency. This involves the arrest of N-methyl-D-aspartate receptor (NMDAR) and α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPAR) currents and paradoxically an increase in γ-aminobutyric acid receptor (GABAR) currents in turtle cortical neurons. In a search for other oxygen-sensitive channels we discovered a Ca(2+)-activated K(+) channel (K(Ca)) that exhibited a decrease in open time in response to anoxia. Single-channel recordings of K(Ca) activity were obtained in cell-attached and excised inside-out patch configurations from neurons in cortical brain sheets bathed in either normoxic or anoxic artificial cerebrospinal fluid (aCSF). The channel has a slope conductance of 223pS, is activated in response to membrane depolarization, and is controlled in a reversible manner by free [Ca(2+)] at the intracellular membrane surface. In the excised patch configuration anoxia had no effect on K(Ca) channel open probability (P(open)); however, in cell-attached mode, there was a reversible fivefold reduction in P(open) (from 0.5 ± 0.05 to 0.1 ± 0.03) in response to 30-min anoxia. The inclusion of the potent protein kinase C (PKC) inhibitor chelerythrine prevented the anoxia-mediated decrease in P(open) while drip application of a phorbol ester PKC activator decreased P(open) during normoxia (from normoxic 0.4 ± 0.05 to phorbol-12-myristate-13-acetate (PMA) 0.1 ± 0.02). Anoxia results in a slight depolarization of turtle pyramidal neurons (∼8 mV) and an increase in cytosolic [Ca(2+)]; therefore, K(Ca) arrest is likely important to prevent Ca(2+) activation during anoxia and to reduce the energetic cost of maintaining ion gradients. We conclude that turtle pyramidal cell Ca(2+)-activated K(+) channels are oxygen-sensitive channels regulated by cytosolic factors and are likely the reptilian analog of the mammalian large conductance Ca(2+)-activated K(+) channels (BK channels).

First aid kit for hypoxic survival: sensors and strategies.

Survival success under conditions of acute oxygen deprivation depends on efficiency of the central and peripheral chemoreception, optimization of oxygen extraction from the hypoxic environment and its delivery to the periphery, and adjustments of energy production and consumption. This article uses a comparative approach to assess the efficiency of adaptive strategies used by anoxia-tolerant and hypoxia-sensitive species to support survival during the first minutes to 1 h of oxygen deprivation. An aquatic environment is much more demanding in terms of diurnal and seasonal variations of the ambient oxygen availability from anoxia to hyperoxia than is an air environment. Therefore, fishes and aquatic turtles have developed a number of adaptive responses, which are lacking in most of the terrestrial mammals, to cope with these extreme conditions. These include efficient central and peripheral chemoreception, acute changes in respiratory rate and amplitude, and acute increase of the gas-exchange interface. A special set of adaptive mechanisms are engaged in reduction of the energy expenditure of the major oxygen-consuming organs: the brain and the heart. Both reduction of ATP consumption and a switch to alterative energy sources contribute to the maintenance of ATP and ion balance in hypoxia-tolerant animals. Hypoxia and hyperoxia are conditions favoring development of oxidative stress. Efficient protection from oxidation in anoxia-tolerant species includes reduction in the glutamate levels in the brain, stabilization of the mitochondrial function, and maintenance of nitric oxide production under conditions of oxygen deprivation. We give an overview of the current state of knowledge on some selected molecular and cellular acute adaptive mechanisms. These include the mechanisms of chemoreception in adult and neonatal mammals and in fishes, acute metabolic adaptive responses in the brain, and the role of nitrite in the preservation of heart function under hypoxic conditions.

Anoxic suppression of Na(+)-K(+)-ATPase and constant membrane potential in hepatocytes: support for channel arrest.

The maintenance of ion gradients across the plasma membrane by the Na(+)-K(+)-ATPase has been shown to utilize a large fraction of the total cellular energy demand. In view of the importance of ion gradients to cellular function, and the remarkable anoxia tolerance of Chrysemys picta bellii (western painted turtle) and hepatocytes isolated from this species, it was of interest to determine if in response to anoxia 1) ion gradients were maintained and 2) if the activity of the plasma membrane Na(+)-K(+)-ATPase changed to aid in ion gradient maintenance. From normoxic hepatocyte suspensions the ouabain-inhibitable 86Rb+ uptake (a measure of Na(+)-K(+)-ATPase activity) was determined, and the rate of ATP utilization was 19.1 mumol ATP.g cells-1.h-1 or 28% of the total normoxic cellular ATP turnover. In response to anoxic incubation the activity of the pump decreased by 75% to 4.8 mumol ATP.g cells-1.h-1 and this comprised 74% of the total anoxic ATP turnover. Presently, it is not known whether the observed reduction in Na(+)-K(+)-ATPase activity is regulated by 1) allosteric modification, 2) endocytosis from the membrane, or 3) reduced Na+ influx. Plasma membrane potential was measured during anoxia, using the distribution of 36Cl-, and was not significantly different from the normoxic measurement, -30.6 +/- 3.9 and -31.3 +/- 5.8 mV, respectively. Therefore, the plasma membrane ion gradient is maintained during anoxia, and since the activity of the Na(+)-K(+)-ATPase decreases, the influx of ions must also decrease.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of adenosine in NMDA receptor modulation in the cerebral cortex of an anoxia-tolerant turtle (Chrysemys picta belli).

Accumulation of the neuromodulator adenosine in the anoxia-tolerant turtle brain may play a key role in a protective decrease in excitatory neurotransmission during anoxia. Since excitatory neurotransmission is mediated largely by Ca2+ entry through N-methyl-D-aspartate (NMDA) receptors, we measured the effect of adenosine on NMDA-mediated Ca2+ transients in normoxic and anoxic turtle cerebrocortical sheets. Intracellular [Ca2+] was measured fluorometrically with the Ca2+-sensitive dye Fura-2. Baseline intracellular [Ca2+] and [ATP] were also measured to assess cortical sheet viability and potential toxic effects of NMDA. Baseline [Ca2+] did not change significantly under any condition, ranging from 109 +/- 22 to 187 +/- 26 nmoll-1. Throughout normoxic and 2h anoxic protocols, and after single and multiple NMDA exposures, [ATP] did not change significantly, ranging from 16.0 +/- 1.9 to 25.3 +/- 4.9 nmol ATP mg-1 protein. Adenosine caused a reduction in the normoxic NMDA-mediated increase in [Ca2+] from a control level of 287 +/- 35 to 103 +/- 22 nmoll-1 (64%). This effect is mediated by the A1 receptor since 8-phenyltheophylline (a specific A1 antagonist) effectively blocked the adenosine effect and N6-cyclopentyladenosine (a specific A1 agonist) elicited a similar decrease in the NMDA-mediated response. Cortical sheets exposed to anoxia alone exhibited a 52% decrease in the NMDA-mediated [Ca2+] rise, from 232 +/- 30 to 111 +/- 9 nmoll-1. The addition of adenosine had no further effect and 8-phenyltheophylline did not antagonize the observed decrease. Therefore, the observed down-regulation of NMDA receptor activity during anoxia must involve additional, as yet unknown, mechanisms.

Effects of fructose-1,6-bisphosphate on glutamate release and ATP loss from rat brain slices during hypoxia.

Fructose-1,6-bisphosphate (FBP), an intermediate of glucose metabolism, is neuroprotective in brain hypoxia or ischemia. Because the mechanisms for this protection are not clear, we examined the effects of FBP on two important events in brain ischemia, i.e., loss of ATP and release of the excitatory neurotransmitter glutamate. Glutamate release from cortical brain slices was measured fluorometrically (glutamate dehydrogenase-catalyzed conversion of glutamate to alpha-ketoglutarate) during hypoxia (PO2 15 mm Hg) or hypoxia plus 100 microM cyanide. FBP (3.5 mM, with glucose 20 mM) reduced glutamate release during hypoxia by 55% and during hypoxia/cyanide by 46% (p < 0.005), and prevented a significant fall in [ATP]. [ATP] was maintained in oxygenated glucose-free conditions with 20 but not 3.5 mM FBP, and fell to < 20% of normal with hypoxia. Despite the drop in [ATP], 3.5 or 20 mM FBP without glucose decreased hypoxia-evoked glutamate release. We conclude (1) FBP present without glucose preserves normal [ATP] only when oxygen is available, suggesting limited uptake and metabolism; and (2) FBP decreases hypoxia-evoked glutamate release by processes independent of [ATP]. These results suggest protective actions of FBP that are separate from augmentation of anaerobic energy production, as previously proposed.

Adaptations of vertebrate neurons to hypoxia and anoxia: maintaining critical Ca2+ concentrations.

Down-regulation of ion channel activity ('channel arrest'), which aids in preserving critical ion gradients in concert with greatly diminished energy production, is one important strategy by which anoxia-tolerant neurons adapt to O2 shortage. Channel arrest results in the elimination of action potentials and neurotransmission and also decreases the need for ion transport, which normally requires a large energy expenditure. Important targets of this down-regulation may be channels in which activity would otherwise result in the toxic increases in intracellular [Ca2+] characteristic of anoxia-sensitive mammalian neurons. In turtles, Na+ channels and the Ca2+-permeable ion channel of the N-methyl-d-aspartate (NMDA)-type glutamate receptor undergo down-regulation during anoxia. Inactivation of NMDA receptors during hypoxia occurs by a variety of mechanisms, including alterations in the phosphorylation state of ion channel subunits, Ca2+-dependent second messenger activation, changes in Ca2+-dependent polymerization/depolymerization of actin to postsynaptic receptors and activation of other G-protein-coupled receptors. Release of inhibitory neurotransmitters (e.g. gamma-aminobutyrate) and neuromodulators (e.g. adenosine) into the brain extracellular fluids may play an important role in the down-regulation of these and other types of ion channels.

Acute reduction in whole cell conductance in anoxic turtle brain.

We tested the effect of anoxia, a "mimic" turtle artificial cerebrospinal fluid (aCSF) consisting of high Ca2+ and Mg2+ concentrations and low pH and adenosine perfusions, on whole cell conductance (G(w)) in turtle brain slices using a whole cell voltage-clamp technique. With EGTA in the recording electrode, anoxic or adenosine perfusions did not change Gw significantly (values range between 2.15 +/- 0.24 and 3.24 +/- 0.56 nS). However, perfusion with normoxic or anoxic mimic aCSF significantly decreased Gw. High [Ca2+] (4.0 or 7.8 mM) perfusions alone could reproduce the changes in Gw found with the mimic perfusions. With the removal of EGTA from the recording electrode, Gw decreased significantly during both anoxic and adenosine perfusions. The A1-receptor agonist N6-cyclopentyladenosine reduced Gw in a dose-dependent manner, whereas the A1-receptor specific antagonist 8-cyclopentyl-1,3-dipropylxanthine blocked both the adenosine- and anoxic-mediated changes in Gw. These data suggest a mechanism involving A1-receptor-mediated changes in intracellular [Ca2+] that result in acute changes in Gw with the onset of anoxia.

Adenosine and anoxia reduce N-methyl-D-aspartate receptor open probability in turtle cerebrocortex.

During normoxia, glutamate and the glutamate family of ion channels play a key role in mediating rapid excitatory synaptic transmission in the central nervous system. However, during hypoxia, intracellular [Ca2+] increases to neurotoxic levels, mediated largely by the N-methyl-D-aspartate (NMDA) subfamily of glutamate receptors. Adenosine has been shown to decrease the magnitude of the hypoxia-induced increase in [Ca2+]i in mammalian brain slices, delaying tissue injury. Turtle brain is remarkably tolerant of anoxia, maintaining a pre-anoxic [Ca2+]i while cerebral adenosine levels increase 12-fold. Employing cell-attached single-channel patch-clamp techniques, we studied the effect of adenosine (200 micromol l-1) and anoxia on NMDA receptor open probability (Popen) and current amplitude. After 60 min of anoxic perfusion, channel Popen decreased by 65 % (from 6.8+/-1.6 to 2.4+/-0.8 %) an effect that could also be achieved with a normoxic perfusion of 200 micromol l-1 adenosine (Popen decreased from 5.8+/-1.1 to 2.3+/-1.2 %). The inclusion of 10 micromol l-1 8-phenyltheophylline, an A1 receptor blocker, prevented the adenosine- and anoxia-induced decrease in Popen. Mean single-channel current amplitude remained at approximately 2.7+/-0.23 pA under all experimental conditions. To determine whether a change in the membrane potential could be part of the mechanism by which Popen decreases, membrane and threshold potential were measured following each experiment. Membrane potential did not change significantly under any condition, ranging from -76.8 to -80.6 mV. Therefore, during anoxia, NMDA receptors cannot be regulated by Mg2+ in a manner dependent on membrane potential. Threshold potentials did decrease significantly following 60 min of anoxic or adenosine perfusion (control -33.3+/-1.9 mV, anoxia -28.4+/-1.5 mV, adenosine -23.4+/-2.8 mV). We conclude that anoxia modulates NMDA receptor activity and that adenosine plays a key role in mediating this change. This is the first direct measurement of ion channel activity in anoxic turtle brain and demonstrates that ion channel regulation is part of the naturally evolved anoxic defence mechanism of this species.

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. METHODS: Glutamate released from cortical brain slices was measured fluorometrically with the glutamate dehydrogenase catalyzed formation of the reduced form of nicotinamide adenine dinucleotide phosphate. Glutamate release was measured in oxygenated (PO2 = 400 mmHg), hypoxic ((PO2 = 20 mmHg), and anoxic ((PO2 = 20 mmHg plus 100 microM NaCN) solutions and with clinical concentrations of anesthetics (halothane 325 microM, enflurane 680 microM, propofol 200 microM, sodium thiopental 50 microM). The source of glutamate released during these stresses was defined with toxins inhibiting N and P type voltage-gated calcium channels, and with calcium-free medium. RESULTS: Glutamate released during hypoxia or anoxia was 1.5 and 5.3 times greater, respectively, than that evoked by depolarization with 30 mM KCl. Hypoxia/anoxia-induced glutamate release was not mediated by synaptic voltage-gated calcium channels, but probably by the reversal of normal uptake mechanisms. Halothane, enflurane, and sodium thiopental, but not propofol, decreased hypoxia-evoked glutamate release by 50-70% (P < 0.05). None of the anesthetics alter basal glutamate release. CONCLUSIONS: The authors conclude that halothane, enflurane, and sodium thiopental but not propofol, at clinical concentrations, decrease extrasynaptic release of L-glutamate during hypoxic stress.

Effects of isoflurane 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 pathophysiologic 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-microns-thick rat cortical brain slices. Temperature was varied to 28, 34, 37, or 39 degrees 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 microM), to ischemia levels of L-glutamate (1.0 mM) or to simulated ischemia (1.0 mM glutamate, 100 microM 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 pathophysiologic events in an in vitro model of simulated cerebral ischemia.

Temperature effects on pH of mitochondria isolated from carp red muscle.

Mitochondria isolated from red muscle of carp (Cyprinus carpio) were used to investigate the effects of temperature and extramitochondrial pH (pHe) on the mitochondrial pH gradient and respiratory properties. Mitochondria from animals acclimated to 10 degrees C were isolated and incubated in KCl-based media with 0.2 mM lauroylcarnitine (C-12) as substrate. Maximal respiratory control ratios (RCR = state 3/state 4) were 16-18 between pH 6.7 and 7.4 at 10 degrees C; RCR values were 9-12 between pH 6.5 and 7.1 at 30 degrees C. Changes in RCR values were due primarily to changes in the state 3 rate (in the presence of ADP). Mitochondrial pH was determined by measuring 5,5-[2-14C]dimethyloxazolidine-2,4-dione distribution, using [14C]sucrose as an extramatrical marker. The pH gradient was inversely related to pHe. At any particular pHe, the mitochondrial pH gradient decreased with increasing temperature. However, if pHe was varied in the same manner that intracellular pH changes with temperature in vivo, the pH gradient was maintained constant at approximately 0.4 U at 10, 20, and 30 degrees C. These data suggest that carp red muscle mitochondria defend an appropriate mitochondrial pH gradient with temperature-induced changes in intracellular pH.