Endogenous GABA _A and GABA _B receptor-mediated electrical suppression is critical to neuronal anoxia tolerance

Abstract

Anoxic insults cause hyperexcitability and cell death in mammalian neurons. Conversely, in anoxia-tolerant turtle brain, spontaneous electrical activity is suppressed by anoxia (i.e., spike arrest; SA) and cell death does not occur. The mechanism(s) of SA is unknown but likely involves GABAergic synaptic transmission, because GABA concentration increases dramatically in anoxic turtle brain. We investigated this possibility in turtle cortical neurons exposed to anoxia and/or GABA A/B receptor (GABAR) modulators. Anoxia increased endogenous slow phasic GABAergic activity, and both anoxia and GABA reversibly induced SA by increasing GABA A Rmediated 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 GABA B R-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 GABA A+B R antagonists underwent seizure-like events, deleterious Ca 2+ 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.western painted turtle | cerebral cortex | channel arrest | pyramidal neurons | natural anesthetic mechanism W hen deprived of oxygen, mammalian neurons are unable to produce sufficient ATP to meet cellular demands (1, 2). As a result, the Na + /K + ATPase (Na + pump) fails and neuronal membrane potential (V m ) becomes unsustainable and anoxic depolarization (AD) follows, causing electrical hyperexcitability, deleterious Ca 2+ influx, and spreading depression in the penumbral region (2, 3). Numerous studies have focused on the role of glutamatergic N-methyl-D-aspartate receptors (NMDARs) in this mechanism, and although NMDAR blockade prevents glutamatergic excitotoxicity (4), it does not prevent AD-mediated injury or postinsult apoptotic cell death (5). Thus, it is not surprising that clinical interventions targeting glutamate receptors alone have been largely ineffective against anoxic or ischemic damage (6), and therefore examination of alternative mechanisms to limit excitability during such insults is necessary.A potential therapeutic alternative to directly antagonizing excitatory pathways is to up-regulate inhibitory mechanisms such as those mediated by γ-aminobutyric acid (GABA), the primary inhibitory neurotransmitter in the mature mammalian CNS (7). GABAergic mechanisms are not strongly recr...

Painted Turtle Cortex is Resistant to anin VitroMimic of the Ischemic Mammalian Penumbra

“…An important component of this tolerance during the transition to IS treatment and reperfusion is an increase in inhibitory GABAergic signaling. GABA is released in anoxic turtle brain (Nilsson and Lutz, 1991) and upon binding to postsynaptic GABA A receptors, activates G Cl to a degree sufficient to dominate G w , and thus clamp V m at E GABA (Pamenter et al, 2011). As a result, excitatory inputs are neutralized by a ready flux of opposing charge mediated by Cl À , which dampens excitatory depolarization via a shunt-like mechanism.…”

“…Cortical slice dissection and patch-clamp recording methods are described elsewhere (Pamenter et al, 2011). Briefly, cortical sheets were dissected and placed in a bath with flow-through perfusion system.…”

“…Briefly, cortical sheets were dissected and placed in a bath with flow-through perfusion system. Neurons were perfused with (1) ACSF (in mmol/L: K + 2.6, Na + 107, Cl À 113, Ca 2 + 1.2, Mg 2 + 1.0, NaH 2 PO 4 2.0, NaHCO 3 26.5, glucose 10, imidazole 5 (280 to 290 mOsm, pH 7.4, 21% O 2 , 5% CO 2 , balance N 2 ); Pamenter et al, 2011) at 41C, (2) ACSF at 241C, or (3) an ischemic penumbral perfusate mimic (IS, in mmol/L: K + 64, Na + 51, Cl À 77.5, Ca 2 + 0.13, Mg 2 + 1.5, glucose 3.0, glutamate 0.1 (315 mOsM, pH 6.5, 1.5% O 2 , 15% CO 2 , balance N 2 ); Yao et al, 2007) at 241C; alone or with pharmacological modifiers as specified in Results section. In some experiments, cortical sheets were treated with ACSF containing high [K + ] or with IS containing normal [K + ] (2.6 mmol/L).…”

“…At rest, neuronal G w was 3.9±0.2 nS, but increased > 3-fold to 14.6 ± 1.3 nS during IS perfusion (n = 31, Figures 2A to 2C); and G w recovered to pre-IS control levels on reperfusion (4.8±0.5 nS). We have previously shown that 2 mmol/L GABA administration induces similar-magnitude changes to G w in turtle neurons (Pamenter et al, 2011); therefore, in separate experiments we treated cortical sheets with GABA during an ACSF to IS transition with recovery. In these experiments, G w in neurons pretreated for 15 seconds with 2 mmol/L GABA was 15.1 ± 1.7 nS in normoxic ACSF and 17.1 ± 2.4 nS in IS (n = 14, Figure 2A).…”

“…In anoxic turtle brain, the conductance and expression of ion channels, in particular excitatory glutamatergic receptors, are rapidly and reversibly decreased (Bickler et al, 2000;, thereby reducing ionic flux and opposing excitatory depolarization. Concomitantly, g-amino butyric acid (GABA), the primary inhibitory neurotransmitter in adult mammalian central nervous system (Krnjevic, 1997), increases > 80-fold in anoxic turtle brain (Nilsson and Lutz, 1991), which activates a dominant conductance to Cl À ions, clamping neuronal membrane potential (V m ) near the Cl À ion reversal potential (E Cl or E GABA ) and opposing excitatory depolarization via a shunt-like inhibitory mechanism (i.e., spike arrest; Pamenter et al, 2011). Together, these neuroprotective mechanisms act to decrease neuronal excitability, ionic flux, and compensatory ion pump activity, thus preserving ATP and enabling neuronal survival during prolonged anoxia (Buck et al, 1998;Hylland et al, 1997;Pamenter et al, 2011).…”