Hypoxia tolerance in the vertebrate brain often involves chemical modulators that arrest neuronal activity to conserve energy. However, in intact networks, it can be difficult to determine whether hypoxia triggers modulators to stop activity in a protective manner or whether activity stops because rates of ATP synthesis are insufficient to support network function. Here, we assessed the extent to which neuromodulation or metabolic limitations arrest activity in the respiratory network of bullfrogs-a circuit that survives moderate periods of oxygen deprivation, presumably, by activating an inhibitory noradrenergic pathway. We confirmed that hypoxia and norepinephrine (NE) reduce network output, consistent with the view that hypoxia may cause the release of NE to inhibit activity. However, these responses differed qualitatively; hypoxia, but not NE, elicited a large motor burst and silenced the network. The stereotyped response to hypoxia persisted in the presence of both NE and an adrenergic receptor blocker that eliminates sensitivity to NE, indicating that noradrenergic signaling does not cause the arrest. Pharmacological inhibition of glycolysis and mitochondrial respiration recapitulated all features of hypoxia, implying that reduced ATP synthesis underlies the effects of hypoxia on network activity. Finally, activating modulatory mechanisms that dampen neuronal excitability when ATP falls, KATP channels and AMP-dependent protein kinase, did not resemble the hypoxic response. These results suggest energy failure- rather than inhibitory modulation- silences the respiratory network during hypoxia and emphasize the need to account for metabolic limitations before concluding that modulators arrest activity as an adaptation for energy conservation in the nervous system.
Lactate ion sensing has emerged as a process that regulates ventilation during metabolic challenges. Most work has focused on peripheral sensing of lactate for the control of breathing. However, lactate also rises in the central nervous system (CNS) during disturbances to blood gas homeostasis and exercise. Using an amphibian model, we recently showed that lactate ions, independently of pH and pyruvate metabolism, act directly in the brainstem to increase respiratory‐related motor outflow. This response had a long washout time and corresponded with potentiated excitatory synaptic strength of respiratory motoneurons. Thus, we tested the hypothesis that lactate ions enhance respiratory output using cellular mechanisms associated with long‐term synaptic plasticity within motoneurons. In this study, we confirm that 2 mM sodium lactate, but not sodium pyruvate, increases respiratory motor output in brainstem‐spinal cord preparations, persisting for 2 h upon the removal of lactate. Lactate also led to prolonged increases in the amplitude of AMPA‐glutamate receptor (AMPAR) currents in individual motoneurons from brainstem slices. Both motor facilitation and AMPAR potentiation by lactate required classic effectors of synaptic plasticity, L‐type Ca2+ channels and NMDA receptors, as part of the transduction process but did not correspond with increased expression of immediate‐early genes often associated with activity‐dependent neuronal plasticity. Altogether these results show that lactate ions enhance respiratory motor output by inducing conserved mechanisms of synaptic plasticity and suggest a new mechanism that may contribute to coupling ventilation to metabolic demands in vertebrates. Key points Lactate ions, independently of pH and metabolism, induce long‐term increases in respiratory‐related motor outflow in American bullfrogs. Lactate triggers a persistent increase in strength of AMPA‐glutamatergic synapses onto respiratory motor neurons. Long‐term plasticity of motor output and synaptic strength by lactate involves L‐type Ca2+ channels and NMDA‐receptors as part of the transduction process. Enhanced AMPA receptor function in response to lactate in the intact network is causal for motor plasticity. In sum, well‐conserved synaptic plasticity mechanisms couple the brainstem lactate ion concentration to respiratory motor drive in vertebrates.
Brain energy stress leads to neuronal hyperexcitability followed by a rapid loss of function and cell death. In contrast, the frog brain switches into a state of extreme metabolic resilience that allows them to maintain motor function during hypoxia as they emerge from hibernation. NMDA receptors (NMDARs) are Ca2+-permeable glutamate receptors that typically drive the loss of homeostasis during hypoxia. Therefore, we hypothesized that hibernation leads to plasticity that reduces the role of NMDARs within neural networks to improve function during energy stress. To test this, we assessed a circuit with a large involvement of NMDAR synapses, the brainstem respiratory network of female bullfrogs,Lithobates catesbieanus. Contrary to our expectations, hibernation did not alter the role of NMDARs in generating network output, nor did it affect the amplitude, kinetics, and hypoxia sensitivity of NMDAR currents. Instead, hibernation strongly reduced NMDAR Ca2+permeability and enhanced desensitization during repetitive stimulation, with shifts in mRNA copy number for NMDAR subunits that modify receptor function. Under severe hypoxia, the normal NMDAR profile caused network hyperexcitability within minutes, which was mitigated by blocking NMDARs. After hibernation, the modified complement of NMDARs protected against hyperexcitability, as disordered output did not occur for at least one hour in hypoxia. These findings uncover state-dependence in the plasticity of NMDARs, whereby distinct changes to receptor function improve neural performance during energy stress without interfering with its normal role during healthy activity.
The brain of most vertebrates is highly sensitive to hypoxia, whereby pathological activity ensues within minutes of exposure. Like most hypoxia‐intolerant vertebrates, brainstem motor networks of the American bullfrog (Lithobates catesbeianus) exhibit hyperexcitability followed by loss of rhythmic activity in severe hypoxia, which outwardly resembles the “anoxic depolarization” in mammals. However, we recently identified that adult bullfrogs acclimated to an aquatic overwintering environment (cold‐acclimated, CA) have increased tolerance to hypoxia when compared to non‐acclimated controls (warm‐acclimated, WA). This response involved avoidance of hyperexcitability and a nearly 30‐fold increase in time until cessation of rhythmic brainstem motor output (~10 minutes to ~3.5 hours measured at 22 °C) supported by a shift to anaerobic glycolysis (Bueschke et al., Current Biology, accepted for publication). Although glycolysis sustains ATP production, a major question remains; how does the motor network maintain homeostasis with such a dramatic reduction in ATP turnover relative to oxidative phosphorylation? Glutamatergic synapses that use NMDA‐type receptors (NMDARs) are energetically demanding due to their Ca2+permeability. Indeed, NMDARs cause excitotoxicity in ischemic stroke models and contribute to respiratory‐related synaptic transmission onto motoneurons in bullfrogs. Therefore, we hypothesized that NMDAR function is altered or reduced to transform the brainstem into a hypoxia‐tolerant state. To test this hypothesis, we made extracellular cranial nerve recordings from in vitro brainstem preparations and compared the sensitivity of WA and CA to the block of NMDARs with an antagonist, AP5. If NMDAR function is reduced or altered following CA, we expected AP5 to improve motor function in WA, but not CA, brainstems during hypoxia. We first confirmed hypoxia tolerance after simulated overwintering. WA preparations stopped at 18 ± 13 min (mean±S.D.), with 10/11 preparations showing hyperexcitable activity. CA preparations functioned for >60 min in all experiments and 0/8 exhibited hyperexcitable activity (Fisher’s exact test to compare proportions of preparations with hyperexcitable activity, p=0.0001). In contrast to our hypothesis, WA and CA preparations did not show differences in sensitivity to APV for respiratory‐related network variables (time until final burst and reduction in motor burst amplitude), suggesting no change in NMDAR function at respiratory synapses. However, AP5 reduced the probability of preparations exhibiting chaotic, hyperexcitable motor output in WA preparations, (3/8 compared to 10/11 in controls, Fisher’s exact test, p=0.040), while CA preparations had no apparent hyperexcitable motor responses over the entire 60‐minute protocol with or without AP5. Thus, activation of non‐respiratory NMDARs contributes to motor hyperexcitability during hypoxia, suggesting that extrasynaptic NMDAR function is reduced or altered in CA animals to constrain motor excitability during hypoxia. Ongoing experime...
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