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

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Environmental remodelling of GABAergic and glutamatergic neurotransmission: Rise of the anoxia-tolerant turtle brain D.W. Hogg, P.J. Hawrysh, L.T. Buck n University of Toronto, Cell & Systems Biology, 25 Harbord St, Toronto, Ontario, Canada M5S 3G5

ar t ic l e i nf o

Keywords: GABA shunting inhibition Spike arrest Channel arrest Mitochondria Reactive oxygen species Mitochondrial permeability transition pore Calcium

a b s t r a c t 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. Crown Copyright & 2014 Published by Elsevier Ltd. All rights reserved.

1. Introduction Earth's changing environment has been a major evolutionary force shaping the diversity of species both in the past and present. From an evolutionary perspective, major climactic events such as periods of glaciation have forced organisms to adapt to shifting environmental conditions and, therefore, are responsible for driving the remodelling of physiological systems. Turtles are one of the oldest living vertebrates, with fossilized remains dating back over 220 million years (Li et al., 2008; Reisz and Head, 2008). They have successfully colonized marine, freshwater, and terrestrial environments demonstrating how incredibly adaptive they are as a species. To enable survival across this broad range of environmental niches, turtles have evolved many specialized physiological adaptations: one particularly remarkable adaptation in freshwater painted turtles of the genus Trachemys and Chrysemys is the ability to survive long-term anoxia while overwintering under ice covered water bodies (Bickler and Buck, 2007). This adaptation is of particular interest to the field of neurobiology (e.g., stroke tolerance and anesthesia) due to potential therapies that could result from unravelling the physiological mechanisms that endow tolerance to low oxygen. From a comparative perspective, studying anoxia-tolerant turtles gives us the opportunity to understand how a brain, an organ extremely sensitive to reduced oxygen availability in mammals, can function without oxygen for long

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periods. This review will focus on the physiological mechanisms that mediate neuroprotection from anoxic stress in brain.

1.1. Metabolic depression as a strategy for surviving long-term anoxia Painted turtles (i.e., Chrysemys picta) can overwinter for more than 4 months while buried in anoxic mud at the bottom of ice covered lakes and ponds (Jackson, 2000; Ultsch, 1989). Typical overwintering temperatures reach
3 1C and since turtles are ectothermic their internal temperature decreases along with their environment. This reduces the rate of enzyme-catalyzed reactions, resulting in a reduction in adenosine triphosphate (ATP) consumption and prolongation of anoxic survival. Indeed, in dived turtles, increasing temperature to 20 1C increases metabolic rate and glycogen consumption and shortens anoxic survival time by
200 times (Herbert and Jackson, 1985; Jackson, 2002). While many of the experiments discussed here were performed at room temperature (20–22 1C) the turtle is still able to survive anoxia 1000– 10,000 times longer than a typical vertebrate after taking Q10 effects into consideration, (Bickler and Buck, 2007; Jackson, 2002; Nilsson and Lutz, 2004). Such an example of metabolic depression indicates that turtles have evolved additional protective mechanisms to survive environmentally-induced anoxic stress. To survive under anaerobic conditions, turtles must maintain ATP turnover, i.e. the balance between ATP production and consumption. Anoxia-tolerant turtles can achieve this because they

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Please cite this article as: Hogg, D.W., et al., Environmental remodelling of GABAergic and glutamatergic neurotransmission: Rise of the anoxia-tolerant turtle brain. J. Thermal Biol. (2014), http://dx.doi.org/10.1016/j.jtherbio.2014.01.003i

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are facultative anaerobes that have the capacity to generate ATP via aerobic respiration when oxygen is available or to circumvent aerobic pathways during oxygen deprivation to produce ATP via alternative anaerobic pathways. To provide sufficient substrate for long-term anoxic survival, turtles amass large amounts of glycogen in the liver (
900 mmol g  1 wet weight liver), which is ten times the capacity of the rat (Hochachka and Somero, 1984). To preserve glycogen and prolong anoxic survival, turtles undergo a large-scale reversible reduction in cellular energy turnover to levels sustainable by anaerobic energy production alone, a process termed metabolic arrest (Hochachka, 1986). The metabolic arrest hypothesis proposes that during periods of hypoxia and/or hypothermia, ATP consumption must accompany decreased ATP production to maintain substrate stores and ionic homeostasis. Typical physiological responses to decreased partial pressure of O2 (pO2) in turtle include: a decrease in heart rate from 30 beats/min at 20 1C to 1 beat/10 min at 3 1C (Herbert and Jackson, 1985), and a greater than 90% decrease in heat output or metabolic rate in hepatocytes (Buck et al., 1993b), brain (Doll et al., 1994) and the whole animal (Jackson, 1968). In addition, in studies from turtle hepatocytes there is suppression of non-essential energy consuming processes, including protein turnover (
90%) and urea synthesis (
74%), to conserve glycolytic substrate stores (Land et al., 1993). Under anoxic conditions, ATP production via glycolysis results in increases in blood lactate from 1 to 200 mmol l  1 and decreases in pH from 8 to o7 (Jackson, 2002). To withstand increases in blood and tissue [H þ ], the turtle has evolved an elevated H þ buffering capacity, including a unique shell buffering system that releases calcium (Ca2þ ) and magnesium carbonates to buffer and provide counter ions to lactic acid and to store lactate in the shell that is released during recovery (Jackson, 2004; Warren and Jackson, 2008). 1.2. Channel arrest as a strategy for surviving long-term anoxia Although mammals and reptiles have significantly divergent evolutionary histories, they have functionally comparable neurological physiology, at least in terms of action potential (AP) generation, neurotransmitters, receptors, and ion channels (Kriegstein and Connors, 1986). As such, the relative energetic requirements of a turtle brain are likely similar to that of a mammalian brain, which requires approximately 20% of the body's energetic demands (Yu et al., 2012). In brain, it is estimated that approximately half of the total energy consumption following AP generation is used to re-establish ion concentration gradients; primarily through the action of the Na þ /K þ ATPase (Howarth et al., 2012; Yu et al., 2012). This is essential for the maintenance of ion homeostasis and membrane potential (Vm), which is required for repetitive AP generation and efficient neuronal communication. In addition, the Na þ gradient determined by Na þ /K þ ATPase activity is indirectly responsible for ion-coupled transport of a variety of ions and molecules, including neurotransmitters such as glutamate (Kanner, 1983; Kaplan, 2002). The high metabolic demand of excitatory signaling can be supported by oxidative phosphorylation; however, anaerobic glycolysis, which only produces 10% of the aerobic ATP supply, is insufficient. When a mammalian brain becomes anoxic (i.e., cerebral stroke and suffocation) and ATP supply falls, ATP-driven ion transport (e.g., Na þ / K þ ATPase) fails resulting in anoxic depolarization, excessive glutamate release, and excitotoxic cell death (ECD) (Hansen, 1985). This does not occur in anoxic turtle brain, where Vm is maintained and cellular excitability is decreased (Pamenter et al., 2011). Maintenance of ionic gradients is particularly important in turtle, as evidenced by studies of Na þ /K þ ATPase activity in turtle liver, in which ion pumping was determined to consume 30% of the total ATP turnover during normoxia and up to 75% of total ATP turnover during anoxia (Buck and Hochachka, 1993a). This

suggests that in anoxic brain, the high rate of ATP consumption by Na þ pumping following AP generation would be an important area in which energy can be saved. One mechanism to reduce the cost of maintaining electrochemical gradients is a decrease in ion movement through ion channels, termed “channel arrest” (Hochachka, 1986). Evidence supporting channel arrest from turtle includes, a 42% decrease in voltage-gated Na þ channel density as determined by [3H]brevetoxin-binding assays (Pseudemys scripta) (Perez-Pinzon et al., 1992b) and a 70% decrease in K þ leakage during anoxia, suggesting inhibition of K þ channels (Trachemys scripta) (Chih et al., 1989; Pek and Lutz, 1997). Evidence for channel arrest from our laboratory includes decreases in: glutamate receptor currents (Pamenter et al., 2008b, Shin and Buck, 2003) and more recently, calcium-activated potassium channel (KCa) currents (Rodgers-Garlick et al., 2013) which will both be discussed in detail below. 1.3. Anoxic regulation of NMDA and AMPA receptors In mammalian brain, a hallmark of anoxic and energetic stress is excitotoxic cell death (ECD), a deleterious sequence of events initiated by failure of ATP-powered ionic pumps resulting in membrane depolarization and hyperexcitability (Choi, 1992; Lipton, 1999). Increased activity elevates extracellular glutamate concentrations to toxic levels within minutes of the onset of anoxia and ultimately results in necrotic and apoptotic cell death. In turtle brain, extracellular glutamate is maintained at basal levels for at least 5 h of anoxia which prevents over-activation of glutamatergic receptors (Nilsson and Lutz, 1991). This is achieved through inhibition of activity-dependent vesicular glutamate release and the maintenance of glutamate uptake transporters early in anoxia (0–1.5 h) (Thompson et al., 2007). In addition to decreased glutamate release, postsynaptic glutamate receptor activity is also decreased. In pyramidal neurons of the dorsal cortex, anoxia leads to a 65% decrease in N-methyl-D-aspartate (NMDA) receptor open probability (Popen) after 60 min of anoxia (Buck and Bickler, 1998a). Whole-cell NMDA receptor currents decrease by 45–65% following only 30 min of anoxia (Pamenter et al., 2008a, 2008c; Shin and Buck, 2003) and a reduction in whole-cell α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor currents by as much as 50–60% (Pamenter et al., 2008b; Zivkovic and Buck, 2010). One possibility for the acute reduction in NMDA receptor was receptor internalization; however, the relative abundance of the NR1 subunit in turtle neuronal membranes remains unchanged until after 3 days of anoxia, suggesting that regulation of NMDA receptors by subunit expression occurs only during long-term anoxia (Bickler et al., 2000). Silencing of NMDA receptors during short-term anoxia was co-incident with an approximately 35% increase in intracellular [Ca2 þ ] ([Ca2 þ ]i) (Bickler, 1998). The modest rise in [Ca2 þ ]i is nonlethal to the cell and is necessary for AMPA and NMDA receptor silencing during anoxia, as chelation of Ca2 þ with BAPTA abolishes the anoxia-mediated reduction in whole-cell AMPA/NMDA receptor currents (Shin et al., 2005; Zivkovic and Buck, 2010). Furthermore, anoxic turtle neurons experience an elevation in [Ca2 þ ]i even in the absence of extracellular [Ca2 þ ], indicating that Ca2 þ originates from an intracellular source (Hawrysh and Buck, 2013; Pamenter et al., 2008c). The two main intracellular stores of Ca2 þ are mitochondria and the endoplasmic reticulum (ER). It is unlikely that Ca2 þ originating from the ER contributes to the reduction in AMPA/NMDA receptor currents as inhibition of ryanodine receptors and the ER Ca2 þ ATPase with ryanodine and thapsigargin, respectively, had no effect on the anoxia-mediated reduction in NMDA receptors (Pamenter et al., 2008c). Based on this information, we suspect that mitochondria are the source of the anoxic rise in [Ca2 þ ]i.

Please cite this article as: Hogg, D.W., et al., Environmental remodelling of GABAergic and glutamatergic neurotransmission: Rise of the anoxia-tolerant turtle brain. J. Thermal Biol. (2014), http://dx.doi.org/10.1016/j.jtherbio.2014.01.003i

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2. Mitochondrial Ca2 þ release regulates glutamatergic receptors during anoxia 2.1. Role of the mitochondrial KATP channel in anoxia-mediated Ca2 þ release As the central site of ATP production and apoptotic initiation, mitochondria are ideal sensors of cellular oxygen levels, metabolite supply, and cellular health. Although cellular ATP remains relatively stable in anoxic turtle neurons (Buck et al., 1998b), it is possible that there are profound regional decreases in [ATP] in areas of high energetic demand which would make ATP a signaling molecule during an energy crisis. Such reasoning would explain the neuroprotective effects attributed to ATP-sensitive potassium (KATP) channels, which are activated in response to lowered [ATP]. Typically, KATP channels are found on the plasma membrane and are composed of four sulfonylurea receptor (SUR) subunits and four inward-rectifier potassium ion channel (Kir6.x) subunits; however, it has been suggested that there also exists a mitochondrial receptor isoform. The proposed existence of the mitochondrial KATP (mKATP) channel is the result of numerous studies including: a singlechannel patch-recording study of ATP-sensitive K þ currents in mitoplasts (Inoue et al., 1991), isolation of mitochondrial protein complexes of similar weight to plasmalemmal KATP channels (Paucek et al., 1992), and mitochondrial matrix swelling observed in the presence of the mKATP channel-specific activator diazoxide (Rousou et al., 2004). However, current molecular evidence of the structure of the mKATP channel has not confidently identified KATP subunits nor has it found any protein-protein interactions between SUR and Kir6.x subunits in the mitochondrial membrane (Hanley and Daut, 2005). Despite the lack of structural evidence, it would be short-sighted to ignore any information pertaining to the physiological relevance of the mKATP channel. The significance of mKATP channels in turtle neurons was first demonstrated by an increase in [Ca2 þ ]i in response to channel activation with diazoxide (Pamenter et al., 2008c). This rise in [Ca2 þ ]i is responsible for the anoxiamediated reduction in glutamatergic currents, as diazoxide treatment also resulted in a 50–60% reduction in whole-cell NMDA/ AMPA receptor currents that was abolished by chelating [Ca2 þ ]i with 1,2-bis(o-aminophenoxy) ethane-N,N,N0 ,N0 -tetraacetic acid (BAPTA) (Pamenter et al., 2008c; Zivkovic and Buck, 2010). Conversely, inhibition of mKATP channels with the specific channel blocker 5-hydroxydecanoic acid (5-HD) prevented the anoxic rise in [Ca2 þ ]i and down-regulation of NMDA/AMPA receptors, demonstrating their importance in the channel arrest mechanism (Pamenter et al., 2008c; Zivkovic and Buck, 2010). Opening of mKATP channels provides favorable conditions for mitochondrial Ca2 þ efflux by depolarizing the mitochondrial membrane potential (Ψm), as evidenced by an increase in fluorescence of the Ψmsensitive dye rhodamine-123 during anoxia or diazoxide application (Hawrysh and Buck, 2013). 2.2. Maintenance of Ψm through complex V activity It is important to mention that inhibition of mKATP channels following anoxia-mediated Ψm depolarization results in a return of Ψm to normoxic baseline levels, suggesting that mKATP channels are constitutively open during anoxic conditions (Hawrysh and Buck, 2013). While such a prolonged permeability would indicate that the Ψm is prone to collapse, it is in fact only mildly depolarized to a new set-point since treatment with carbonyl cyanide4-(trifluoromethoxy)phenylhydrazone (FCCP) (H þ ionophore and uncoupler of oxidative phosphorylation in mitochondria) following anoxia can further depolarize Ψm (Hawrysh and Buck, 2013). This new Ψm set-point is maintained by reversal of the F1F0– ATPase (Complex V), as treatment with its inhibitor oligomycin

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resulted in Ψm depolarization to a level comparable to that following FCCP treatment (Hawrysh and Buck, 2013). Preventing Ψm collapse by complex V reversal is energetically costly however, as it hydrolyzes ATP to pump protons out of the mitochondrial matrix. It is therefore imperative for anoxia-tolerant species to adapt a strategy to avoid using excessive ATP while maintaining Ψm. Some examples of such a strategy may include increasing F1 inhibitory subunit activity, limiting ATP transport into the mitochondria via the adenine nucleotide translocase, or modifying the Km of complex V for ATP (St-Pierre et al., 2000). A reduction in complex V activity has been demonstrated in several anoxiatolerant organisms such as the fresh-water turtle T. scripta, the common frog Rana temporaria, and the diapausing embryo of the annual killifish Austrofundulus limnaeus (Duerr and Podrabsky, 2010; Galli et al., 2013; St-Pierre et al., 2000). The level of complex V activity in the western painted turtle during anoxia is likely comparable to that of T. scripta, which experiences an 85% reduction in complex V activity after two weeks of anoxia (Galli et al., 2013). However it is probable that this still translates to a significant amount of anaerobically-derived ATP, as complex V activity accounts for approximately 9% of ATP turnover during anoxia in skeletal muscle of the anoxia-tolerant frog (St-Pierre et al., 2000). This would place Ψm regulation in the anoxic mitochondrion at a high priority, since maintenance of Ψm is required for protein import and proper mitochondrial function while Ψm collapse induces irreversible cell death (Kroemer et al., 1998; Wiedemann et al., 2004). Cell death in the anoxic turtle brain is avoided as indicated by propidium iodide cell death assays that show membrane permeabilization does not occur after 4 h of anoxia or even 24 h of ischemia (Pamenter et al., 2011, 2012) and it is likely that this is due to the turtle's ability to defend Ψm. 2.3. Role of the mPTP in anoxia-mediated Ca2 þ release In anoxia-intolerant rat mitochondria, elevated extra-mitochondrial Ca2 þ induces opening of the mitochondrial permeability transition pore (mPTP), mitochondrial swelling, and cytochrome c release. Conversely, in anoxia-tolerant organisms such as the embryos of Artemia franciscana, a similar increase in extra-mitochondrial Ca2þ activates a modified form of the mPTP and does not induce mitochondrial permeability (Menze et al., 2005). This “recalcitrant mPTP” has been suggested to be a low-conductance form of the mPTP that is activated following decreases in oxygen as an emergency mechanism to release accumulated Ca2þ and decrease the rate of reactive oxygen species (ROS) formation by lowering Ψm (Huser and Blatter, 1999). A reduction in ROS would prevent prolonged, highconductance openings of the mPTP that would otherwise lead to cell death, as increased levels of ROS are associated with high-conductance opening of the mPTP and apoptosis (Brookes et al., 2004). The lowconductance form of the mPTP is only permeable to small ions such as Ca2 þ but impermeable to larger compounds, including, apoptosisinducing-factor or cytochrome c. This allows molecules with an atomic mass up to 300 Dalton (Da) to cross the mitochondrial membranes rather than the 1500 Da permitted via the high-conductance, apotosisinducing form (Haworth and Hunter, 1979a, 1979b; Ichas and Mazat, 1998). While it has been firmly established that anoxic turtle mitochondria release Ca2 þ , it has only recently been demonstrated that a form of the mPTP is involved in Ca2 þ efflux. Evidence supporting a role for mPTP opening during anoxia in turtle mitochondria includes attenuation of Ca2 þ release and prevention of NMDA receptor silencing in the presence of the mPTP inhibitor cyclosporine-A (Hawrysh and Buck, 2013). Pore opening is regulated by several factors; however, the mPTP is particularly sensitive to changes in intracellular/matrix pH. In rat mitochondria, decreases in pH inhibit Ca2 þ movement through the mPTP, with a

Please cite this article as: Hogg, D.W., et al., Environmental remodelling of GABAergic and glutamatergic neurotransmission: Rise of the anoxia-tolerant turtle brain. J. Thermal Biol. (2014), http://dx.doi.org/10.1016/j.jtherbio.2014.01.003i

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50% decrease in Ca2 þ -mediated mitochondrial swelling occurring at a pH of 6.7 (heart) and 7.0 (liver) (Halestrap, 1991). In turtle neurons, 1 h of anoxia shifts intracellular pH from
7.8 to 6.7 (Buck and Bickler 1998a, Buck et al., 1998b), which favors reduced pore opening and supports our hypothesis of mPTP-mediated Ca2 þ release during short-term anoxia. Pore activation can also occur in response to lowered adenylate phosphate concentrations or Ψm depolarization (Bernardi, 1992; Hunter and Haworth, 1979), two conditions that are associated with mKATP channel activation. Our current hypothesis proposes that upon onset of anoxia, local decreases in [ATP] in the mitochondria matrix activates mKATP channels, which results in Ψm depolarization, mPTP opening and Ca2 þ release (Fig. 1A and B). It is likely that the turtle utilizes this recalcitrant form of the mPTP since turtle neurons do not experience cell death during anoxia (Pamenter et al., 2011); however, it is currently unknown if this mPTP can switch to the highconductance form seen in anoxia-intolerant species. Further characterization of the structure and function of this recalcitrant mPTP is required to elucidate its role during anoxia.

3. Anoxia-mediated regulation of oxygen-sensitive Ca2 þ -activated K þ channels 3.1. K þ channels and anoxia-tolerance Re-establishment of Vm following AP generation is due to activation of voltage-sensitive K þ (Kv) channels responsible for fast hyperpolarizing currents and Ca2 þ -activated K þ channels responsible for after-hyperpolarization (AHP) currents (Sah, 1996). There are three general families of Ca2 þ -activated K þ channels identified through both biophysical and pharmacological means: the large-conductance (BK or Maxi-K) channels, intermediate-conductance (IK) channels, and small-conductance (SK) channels (Sah and Faber, 2002). Ca2 þ activated K þ channels are essential for proper electrical signaling and perturbation of these factors can result in hyperexcitability and cell death (Hansen, 1985). Maintenance of Vm and transmembrane [K þ ] gradients is achieved primarily by the Na þ /K þ ATPase which is sensitive to increases in extracellular [K þ ] ([K þ ]e) and intracellular Na þ ([Na þ ]i) (Skou and Esmann, 1992; Therien and Blostein, 2000). Therefore, preventing K þ efflux is one method to conserve ATP under anoxic conditions. In mammal brain, anoxic and ischemic stresses induce Vm depolarization due to failure of the Na þ /K þ ATPase resulting in derangement of Na þ and K þ ionic gradients, increased glutamate release/decreased glutamate reuptake, and excessive electrical activity which further depletes [ATP] (Hansen, 1985; Martin et al., 1994; Satoh et al., 1999). In addition, increases in [Ca2 þ ]i through voltage-gated NMDA receptors and Ca2 þ channels are particularly damaging as Ca2 þ can over-activate a variety of enzymes resulting in cell death. Therefore, a combined decrease in Na þ and K þ channel currents would be beneficial in preventing overactivation of the Na þ pump and result in energy conservation. Indeed, suppression of K þ channels in turtle brain seems to be an important component of its anoxia-tolerant strategy. In anoxic turtle brain, there is a 50% decrease in whole-cell K þ conductance (Chih et al., 1989) resulting in a net decrease in K þ leakage (Pek and Lutz, 1997) and only a small increase in cerebral [K þ ]e from 2.6 to 3.7 mM (Sick et al., 1982), indicating tight regulation of [K þ ] gradients during anoxia. This suggests that K þ channels are inhibited during anoxia in turtle brain which is indicative of ion channel arrest (Pek and Lutz, 1997). K þ channels are a common component of oxygen-sensing machinery in oxygen sensing tissues. In particular, the hypoxic inhibition of largeconductance Ca2 þ -activated BK channels has been the focus of numerous studies investigating oxygen-sensing mechanisms in neuroepithelial bodies (Youngson et al., 1993; Williams et al.,

2004; McCartney et al., 2005), adrenomedullary chromaffin cells (Fearon et al., 2002; Thompson and Nurse, 1998) and carotid body glomus cells (Lopez-Barneo et al., 1988, 1993; Williams et al., 2004). For example, in the carotid body glomus cell, decreasing pO2 inhibits K þ currents, resulting in Vm depolarization and increased cellular excitability, elevated cytosolic Ca2 þ , and neurotransmitter release. This increases firing of afferent fibers which terminate in respiratory centers of the brainstem and leads to increased lung ventilation (Lopez-Barneo et al., 1993). Similar to the BK channels found in oxygen-sensing tissue, there are neuronal BK channels located throughout the mammalian brain that require both Ca2 þ and Vm depolarization for channel activation and are involved in fast afterhyperpolarization (Sah and Faber, 2002).

3.2. Identification of an oxygen-sensitive Ca2 þ -activated K þ channel In search of oxygen-sensitive ion channels in pyramidal neurons of the turtle cerebrocortex, our lab recently identified a largeconductance Ca2 þ -activated K þ channel (KCa) that undergoes a reduction in channel open probability (Popen) following anoxic exposure (Rodgers-Garlick et al., 2013). It has characteristics similar to mammalian BK channels including a large conductance (
223 pS), sensitivity to [Ca2 þ ]i which is dependent on Vm depolarization, and inhibition by the specific BK channel inhibitor iberiotoxin. Patch excision prevented the anoxic reduction in Popen, demonstrating that the channel is not intrinsically oxygensensitive but requires modulation by intracellular second messenger molecules. These findings are supported by studies in mouse neocortical neurons (Liu et al., 1999) and rat carotid body glomus cells (Wyatt and Peers, 1995) where patch excision also prevented hypoxic decreases in KCa channel Popen. In turtle brain, anoxia induces mitochondrial Ψm depolarization and a mild increase in [Ca2 þ ]i from 135 to 185 nM (Bickler, 1998; Pamenter et al., 2008c). The anoxia-mediated mitochondrial Ca2 þ signal initiates channel arrest of NMDA and AMPA receptors through a protein phosphatase 1 and 2A-mediated pathway (Pamenter et al., 2008c; Shin et al., 2005; Zivkovic and Buck, 2010). This indicates that mitochondria function as oxygen sensors and suggests that other Ca2 þ sensitive second messenger pathways may be responsible for regulation of KCa channels. Mammalian BK channels are regulated by a number of cytosolic factors including phosphorylation, gases (oxygen, nitric oxide, and carbon monoxide), pH, and reactive oxygen species (Jaggar et al., 2002; Lang et al., 2000; Liu et al., 1999; Riesco-Fagundo et al., 2001). To further characterize the anoxic regulation of turtle neuronal KCa channels the effect of channel phosphorylation / dephosphorylation was investigated. Two kinases known to regulate BK channel function in mammal neurons are protein kinase C (PKC) which decreases channel Popen, and Ca2 þ /calmodulindependent kinase II (CAMKII) which increases channel Popen (van Welie and du Lac, 2011). To assess the role of PKC in the anoxiamediated channel arrest of KCa channels we modulated PKC activity with the PKC inhibitor chelerythrine during anoxia, and the PKC activator phorbol 12-myristate 13-acetate (PMA) during normoxia. We determined that inhibition of PKC prevented the anoxic decrease in Popen and activation of PKC decreased Popen during normoxia. This provides evidence that PKC activity is enhanced under anoxic conditions and responsible for decreasing KCa channel Popen in turtle pyramidal neurons. This PKC is a diacylglycerol and Ca2 þ activated kinase; and therefore, the anoxia-mediated mitochondrial Ca2 þ release is a likely signal to activate PKC and induce KCa channel phosphorylation and inhibition. To assess the steady state normoxic regulation of KCa channels we inhibited the activity of Ca2 þ /calmodulin-dependent kinase II (CAMKII) by applying the Ca2 þ /calmodulin inhibitor

Please cite this article as: Hogg, D.W., et al., Environmental remodelling of GABAergic and glutamatergic neurotransmission: Rise of the anoxia-tolerant turtle brain. J. Thermal Biol. (2014), http://dx.doi.org/10.1016/j.jtherbio.2014.01.003i

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Fig. 1. Summary of the anoxia-mediated mitochondrial calcium release mechanism that leads to NMDA receptor silencing. (A) In response to a reduction of oxygen, oxidative phosphorylation halts and ATP production is reduced (1). This results in local reductions of [ATP] and opening of mitochondrial ATP-sensitive potassium (mKATP) channels (2). This causes depolarization of the mitochondrial membrane potential (Ψm) (3) and opening of the mitochondrial permeability transition pore (mPTP), which leads to calcium release (4). Potassium conductance through mKATP channels is balanced by the mitochondrial K þ /H þ exchanger, which pumps K þ into the cytosol and H þ into the matrix (5). The influx of H þ is balanced by reversal of the ATP synthase, which pumps H þ out via ATP hydrolysis and prevents mitochondrial Ψm collapse (6). (B) Depolarization of Ψm via activation of mKATP channels results in opening of the mPTP and release of calcium from mitochondrial stores (1–5). This results in a modest rise in intracellular [Ca2 þ ] (7). This results in calcium-mediated activation of PKC (8) which phosphorylates calcium-activated plasmalemmal K þ channels and reduces channel conduction (8). The rise in [Ca2 þ ]i also activates calmodulin, which competitively antagonizes binding of α-actinin-2 to the NMDA receptor, resulting in Ca2 þ -dependent inactivation of NMDA receptors and delocalization from the synapse via dissociation from cytoskeletal elements (9).

calmidazolium. However, this had no effect on KCa channel Popen indicating another kinase may be responsible for the activation of KCa channels. Protein phosphatase 1 and 2A have also been shown to regulate BK channel function (van Welie and du Lac,

2011). To assess if these phosphatases also modulate KCa channel activity the phosphatase 1 and 2A inhibitor okadaic acid was applied under normoxic conditions; however, it did not have an effect on KCa channel Popen indicating another phosphatase may be

Please cite this article as: Hogg, D.W., et al., Environmental remodelling of GABAergic and glutamatergic neurotransmission: Rise of the anoxia-tolerant turtle brain. J. Thermal Biol. (2014), http://dx.doi.org/10.1016/j.jtherbio.2014.01.003i

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responsible for the dephosphorylation of KCa channels in turtle brain. Together these data indicate that mitochondrial Ca2 þ release is at least in part responsible for initiating signaling cascades that result in KCa channel arrest, and points to a common mitochondrial based mechanism to decrease ion leak during anoxia. In anoxic turtle cerebrocortex, channel arrest of glutamate receptors and GABA-mediated spike arrest (see below) decrease AP generation and, therefore, the requirement for KCa channelmediated after-hyperpolarization. On the surface this might suggest that channel arrest of KCa channels may be redundant; however, anoxia depolarizes Vm
8 mV (Pamenter et al., 2011) and since the Ca2 þ sensitivity of KCa channels is voltage dependent, arresting KCa channels may prevent depolarization induced KCa currents. In addition, anoxic increases in [Ca2 þ ]i could directly activate KCa channels; thus, arrest of a KCa channels would be an important protective measure to prevent excessive K þ leak. Another potential mechanism to induce channel arrest in KCa channels during anoxia is a decrease in cytosolic pH. Studies from mouse neocortical neurons show BK channel Popen to decrease by
50% in response to acute changes in pH from 7.0 to 6.5 (Liu et al., 1999). This is very close to the change in intracellular pH observed in anoxic turtle brain sheets measured with 31P nuclear magnetic resonance spectroscopy (Buck et al., 1998b). Overall, an oxygen-sensing mechanism may consist of a decrease in mitochondrial [ATP] due to the lack of oxidative phosphorylation which activates mKATP channels, leading to mitochondrial K þ influx, Ψm depolarization, Ca2 þ efflux to the cytosol, PKC activation, and KCa channel inhibition (Hawrysh and Buck, 2013; Pamenter et al., 2008a; Shin et al., 2005).

4. Anoxic regulation of GABA receptors 4.1. GABA receptors and anoxia-tolerance In turtle brain, anoxic submergence results in an 80-fold elevation in striatal [GABA] (Nilsson and Lutz, 1991), and a 75–95% suppression of whole brain electrical activity (Fernandes et al., 1997; Perez-pinzon et al., 1992a). Anoxic increases in GABA-mediated signaling, termed GABAergic spike arrest, are the primary cause of electrical depression and are essential for anoxia-tolerance in brain (Pamenter et al., 2011). An increase in [GABA] is also found in anoxia-tolerant crucian carp, Carassius carassius (Hylland and Nilsson, 1999), indicating an important role for GABAergic inhibition in anoxic survival. GABA is known to be the primary inhibitory neurotransmitter in the mature mammalian central nervous system (CNS) (Krnjevic, 1997) and synaptic release activates two main types of receptors: (1) type A ionotropic GABA receptors (GABAA receptors) – permeable to Cl  and HCO3 and the primary mediator of fast synaptic inhibition in the brain; and (2) type B metabotropic GABA receptors (GABAB receptors) – Gprotein coupled receptors that activate an inwardly rectifying K þ channel postsynaptically and inhibit a voltage-gated Ca2 þ channel presynaptically (Ben-Ari et al., 2007; Kaila et al., 1993). In adult mammalian brain, extracellular [Cl  ] is much higher than intracellular [Cl  ] ([Cl  ]i) due to the higher relative activity of the Cl  extrusion transporter KCC2 (K þ /Cl  cotransporter) compared to the major Cl  uptake transporter NKCC1 (Na þ /K þ /Cl  cotransporter) (Blaesse et al., 2009). GABAA receptors are approximately four times more permeable to Cl  than to HCO3 (Kaila et al., 1989; Kaila and Voipio, 1987); therefore, the reversal potential for GABA (EGABA) at rest (typically  75 mV) is more depolarized than the Cl  reversal potential (ECl  ; typically 85 mV) due to the influence of the HCO3 reversal potential (EHCO3 ; typically  20 mV) (Kaila et al., 1993; Lambert and Grover, 1995). Mammalian neuronal resting Vm is approximately 70 mV; therefore, GABA binding to GABAA receptors results in Cl  influx down its concentration gradient and hyperpolarization of Vm away from action potential threshold (APth), opposing

excitatory inputs (e.g., glutamatergic excitation). Activation of presynaptic GABAB receptors reduces the release of neurotransmitter through a reduction in Ca2 þ activated vesicle fusion, while activation of postsynaptic GABAB receptors hyperpolarizes Vm towards the K þ reversal potential (
 90 mV) and results in decreased neuronal excitation. 4.2. GABA-mediated spike arrest in pyramidal neurons Glutamatergic pyramidal neurons are the most numerous type of neuron in the cerebrocortex of the turtle, accounting for approximately 75% of excitable cells (Connors and Kriegstein, 1986; Kriegstein and Connors, 1986). Since they are responsible for the primary excitatory output of the cerebrocortex, prevention of overexcitation in these neurons during periods of low oxygen is a priority. Under normoxic conditions, pyramidal neurons exhibit slow phasic inhibitory postsynaptic currents (IPSC) that occur at approximately 10 s intervals (Pamenter et al., 2011). These currents are inhibited by GABAA receptor antagonists (e.g., gabazine and picrotoxin) confirming that they are GABAA receptor-mediated. Anoxic treatment doubles current amplitude above resting normoxic values, indicating that this neuroprotective GABAergic signaling mechanism is oxygensensitive. Inhibition of GABA uptake from the synapse with GABA transporter (GAT) blockers increases current amplitude demonstrating that GABA is endogenously released from presynaptic GABAergic interneurons (Pamenter et al., 2011). Anoxic increases in GABA release or pharmacological drip application of GABA activates postsynaptic GABAA receptors and results in a 40% increase in whole-cell conductance (Gw) (from
5 to 7 nS). Pyramidal neurons have a relatively hyperpolarized Vm (
 85 mV) and an EGABA of
 70 mV, therefore, GABA binding to GABAA receptors results in Cl  efflux and depolarization of Vm to EGABA. Although GABA is depolarizing, it remains inhibitory because it activates a shunting current that maintains Vm (at EGABA) below APth. In spontaneously active pyramidal neurons, anoxia decreases AP frequency (APfreq) by
70% (from 2 to 0.6 Hz) providing direct evidence for decreased excitability (Pamenter et al., 2011). In addition to preventing AP generation, APth also depolarizes from approximately  49 to  31 mV during anoxia (Pamenter et al., 2011). This indicates that there is a decrease in Na þ channel activity or density, a finding that is supported by previous measurements of anoxic decreases in voltage-gated Na þ channels in turtle cerebellum (Perez-Pinzon et al., 1992b). All of the anoxia-induced changes could be reversibly blocked by inclusion of the GABAA receptor inhibitor gabazine with the exception of decreases in APfreq which required the presence of GABAB receptor specific inhibitor-CGP55845. This suggests a presynaptic role for GABAB receptors in anoxia-tolerance through inhibition of excitatory neurotransmitter release (e.g., glutamate), since inhibition of both receptors is required to cause anoxic depolarization and cell death during anoxia. We conclude that both GABAA þ B receptors are necessary for anoxia tolerance.

5. Future directions 5.1. Oxygen-sensitive stellate interneurons The primary inhibitory neurons of the cerebrocortex are GABAergic stellate interneurons. These are the second most abundant type of neuron, and they synapse onto and modulate pyramidal neuron activity (Connors and Kriegstein, 1986; Kriegstein and Connors, 1986). To understand the role of GABA in anoxia tolerance and elucidate the mechanism of GABAergic spike arrest it is essential that we characterize the interaction between these types of neurons. Evidence supporting stellate interneurons as the source of anoxic GABA release is limited due to the low incidence of recording from this type of

Please cite this article as: Hogg, D.W., et al., Environmental remodelling of GABAergic and glutamatergic neurotransmission: Rise of the anoxia-tolerant turtle brain. J. Thermal Biol. (2014), http://dx.doi.org/10.1016/j.jtherbio.2014.01.003i

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neuron. However, preliminary data indicates that stellate interneuron activity (bursts of APs) is increased during anoxia, indicating an important role in preventing over-excitation. The temporal occurrence of these AP bursts matches that of the GABAergic IPSCs in pyramidal neurons suggesting that spike arrest is the result of GABA release from oxygen-sensitive stellate interneurons. Although the mechanism responsible for oxygen-sensitivity has yet to be elucidated this evidence indicates that the stellate interneuron/pyramidal neuron network is an important inhibitory control point for dampening electrical activity during anoxia. 5.2. Decreased [ROS]i as an anoxic signal: impact on GABAergic signaling To elucidate the neuroprotective mechanisms responsible for anoxia-tolerance in turtle brain it is necessary to identify the putative oxygen sensors and their respective modulators. Of particular interest is to understand how increased GABAergic signaling is sensed and transmitted. One potential signal is the decrease in reactive oxygen species (ROS) that occurs following the onset of anoxia. In turtle cerebrocortex, extracellular pO2 reaches
0 mm Hg after 1 h of anoxic submergence at room temperature (22–24 1C) (Jackson, 1968; Pamenter et al., 2007). Oxygen is rapidly metabolized by mitochondria resulting in a decrease in intracellular ROS ([ROS]i). Importantly, ROS-mediated signaling would occur simultaneously throughout the brain; and since this mechanism does not require energy input during a time when cellular ATP is limited it constitutes a metabolically inexpensive redox-sensitive signal to coordinate the down-regulation of energy consuming processes on a broad scale. Decreases in [ROS]i have the potential to modulate cysteine-containing proteins involved in various aspects of neurotransmitter signaling including presynaptic neurotransmitter release, postsynaptic receptors, channels and transporters, and second messenger signaling molecules. Preliminary experiments have found that pharmacological application of ROS scavengers mimic's anoxic increases in GABAergic IPSC's in postsynaptic pyramidal neurons while application of hydrogen peroxide (a ROS compound) prevents IPSCs during anoxia suggesting an important role for ROS in modulating GABAergic signaling in the anoxic turtle cerebrocortex. Future experiments will focus on elucidating this redox-sensitive signaling pathway to better understand the role of ROS on vesicular GABA release and post-synaptic GABA receptor function.

6. Summary To survive seasonal ice cover, freshwater painted turtles have evolved physiological mechanisms to endure long-term anoxia. Of primary importance to anoxic survival is the downregulation of metabolic processes to prevent overconsumption of limited fuel resources. In brain, this includes the downregulation of glutamatergic signaling and the upregulation of GABAergic signaling to decrease electrical activity, and prevent ionic dysregulation and toxic Ca2 þ accumulation. Postsynaptic NMDA and AMPA receptors are downregulated by a unique oxygen-sensitive mitochondrial Ca2 þ release mechanism resulting in a reduction in Na þ and Ca2 þ currents. Ca2 þ is released by opening of the mPTP triggered by decreasing mitochondrial Ψm following mKATP activation by an anoxia-mediated decrease in local [ATP]. Mitochondrial Ψm reaches a new set-point that is maintained by reversal of F1F0 ATP synthase and we speculate that maintenance of Ψm is important for the inhibition of apoptosis. The anoxia-mediated increase in [Ca2 þ ]i may also be responsible for regulation of other ion channels, including KCa channels, which decrease activity following the onset of anoxia. Anoxic increases in GABA release activate postsynaptic GABA receptor currents resulting

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in postsynaptic inhibition and spike arrest. The principle inhibitory neurons in the turtle cerebrocortex are the stellate interneurons, which are the likely source of GABA responsible for GABA-mediated spike arrest. Of particular future interest is the role of ROS in initiating elevated GABA release in response to decreasing oxygen levels.

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Please cite this article as: Hogg, D.W., et al., Environmental remodelling of GABAergic and glutamatergic neurotransmission: Rise of the anoxia-tolerant turtle brain. J. Thermal Biol. (2014), http://dx.doi.org/10.1016/j.jtherbio.2014.01.003i