Hyperbaric Hyperoxia and Normobaric Reoxygenation Increase Excitability and Activate Oxygen-induced Potentiation in CA1 Hippocampal Neurons

Breathing hyperbaric oxygen (HBO) is common practice in hyperbaric and diving medicine. The benefits of breathing HBO, however, are limited by the risk of central nervous system O2 toxicity, which presents as seizures. We tested the hypothesis that excitability increases in CA1 neurons of the rat hippocampal slice (400 μm) over a continuum of hyperoxia that spans normobaric and hyperbaric pressures. Amplitude changes of the orthodromic population spike were used to assess neuronal O2 sensitivity before, during, and following exposure to 0, 0.6, 0.95 (control), 2.84, and 4.54 atmospheres absolute (ATA) O2. Polarographic O2 electrodes were used to measure tissue slice Po2 (PtO2). In 0.95 ATA O2, core PtO2 at 200 μm deep was 115 ± 16 Torr (mean ± SE). Increasing O2 to 2.84 and 4.54 ATA increased core PtO2 to 1,222 ± 77 and 2,037 ± 157 Torr, respectively. HBO increased the orthodromic population spike amplitude and usually induced hyperexcitability (i.e., secondary population spikes) and, in addition, a long-lasting potentiation of the orthodromic population spike that we have termed “oxygen-induced potentiation” (OxIP). Exposure to 0.60 ATA O2 and hypoxia (0.00 ATA) decreased core PtO2 to 84 ± 6 and 20 ± 4 Torr, respectively, and abolished the orthodromic response. Reoxygenation from 0.0 or 0.6 ATA O2, however, usually produced a response similar to that of HBO: hyperexcitability and activation of OxIP. We conclude that CA1 neurons exhibit increased excitability and neural plasticity over a broad range of PtO2, which can be activated by a single, hyperoxic stimulus. We postulate that transient acute hyperoxia stimulus, whether caused by breathing HBO or reoxygenation following hypoxia (e.g., disordered breathing), is a powerful stimulant for orthodromic activity and neural plasticity in the CA1 hippocampus. hyperbaric oxygen (HBO) is breathed during HBO therapy (82) and during underwater diving operations and disabled submarine rescue (41, 61). What limits the use of HBO in each case is the potential risk of central nervous system (CNS) O2 toxicity. The hallmark sign of CNS O2 toxicity is unpredictable tonic clonic seizures, which can begin when neural tissue Po2 (PtO2) increases from the normoxic range (\u3c10–45 Torr) to ∼240 Torr or more (7, 83). One key factor in the pathogenesis of CNS O2 toxicity appears to be the accumulation of reactive oxygen species (ROS) and reactive nitrogen species (RNS). Increased production and accumulation of ROS/RNS leads to oxidation of essential cellular constituents such as enzymes, membrane lipid bilayers, and proteins (19), which in turn, alters intrinsic membrane properties and synaptic transmission (4, 5, 12, 51, 56). Electrophysiological studies in rat brain slices indicate that HBO decreases membrane conductance and stimulates firing rate in neurons of the hippocampus and medulla oblongata (25, 50, 65). The excitatory effect of HBO on neurons is blocked by antioxidants and mimicked by prooxidants, which supports the hypothesis that during hyperoxia, increases in ROS/RNS cause elevated neuronal excitability (65). The exact cellular mechanisms underlying O2-induced seizure (O2 toxicity) are poorly understood because of the paucity of electrophysiological studies that have examined the effects of hyperoxia on neurons (50, 65). By contrast, a vast amount of literature exists on the electrophysiological effects of hypoxia on neurons, which has been reviewed elsewhere (57, 76). The main reason for the lack of information on how hyperoxia affects neurons under in vitro conditions is that electrophysiological studies conducted in brain slices employ a hyperoxic control condition using ∼0.95 atmospheres absolute (ATA) O2; i.e., nutrient medium equilibrated with 95% O2 at barometric pressure (Pb) ≅ 1 ATA (1, 64). Consequently, the only way to study the effects of further hyperoxia (\u3e0.95 ATA O2)1 in the brain slice preparation is to use hyperbaric pressure to produce HBO (25, 50, 65, 66). In addition, the effects of relative hyperoxia on PtO2 and excitability can be studied at normobaric pressure (Pb ≅ 1 ATA) during reoxygenation following acute exposure to an intermediate level of O2 (0.6 ATA) or anoxia (0.0 ATA), as in previous studies (17, 18, 42, 44), which we have termed “normobaric reoxygenation” (NBOreox). In the present study, we used HBO and NBOreox to study the effects of a broad range of hyperoxia on PtO2 and neuronal excitability in the CA1 region of the rat hippocampal tissue slice. We tested the hypothesis that neuronal excitability increases over a broad continuum of PtO2 at normobaric and hyperbaric pressures. Although such an excitatory effect of increasing PtO2 on CA1 electrical activity in the hippocampus might be predicted, to our knowledge, no single study has described the O2 sensitivity of CA1 neurons over a range of PtO2 that spans both normobaric and hyperbaric pressures. Few in vitro studies, in fact, have examined O2 sensitivity of neurons in tissue slices at levels of PtO2 \u3e0.95 ATA O2 (25, 50, 65) or \u3c0.95 ATA but ≥0.20 ATA O2 (33, 87). To accomplish this, we recorded the orthodromic population spike (oPS) from the CA1 neuronal population over a broad range of PtO2 using superfusate equilibrated with 0.00 (hypoxia), 0.60 (intermediate oxygenation), 0.95 (control), and 2.84 and 4.54 ATA O2 (HBO). In an additional set of experiments, PtO2 profiles in 400-μm-thick hippocampal slices were measured under identical conditions of Pb, PtO2, pH, temperature, and artificial cerebral spinal fluid (aCSF) flow rate. Our findings demonstrate that beginning in 0.95 ATA O2, neuronal excitability, in general, increased as PtO2 increased and decreased as PtO2 decreased; however, the greatest sensitivity to O2 manipulation occurred between 0.6 and 0.95 ATA O2. Our results also indicate that a single hyperoxic stimulus delivered at normobaric pressure (NBOreox) or hyperbaric pressure (HBO), initiated from 0.0, 0.6, or 0.95 ATA O2, induces hyperexcitability (i.e., secondary population spikes, sPS) and long-lasting neural plasticity. We have termed this form of neural plasticity “O2-induced potentiation” (OxIP) of the oPS. Our findings further support an earlier report (64) that experimental O2 conditions (95% O2) commonly used with the brain slice preparation (∼400 μm thick) are hyperoxic compared with the normoxic CNS of an air-breathing animal. A second study, described in the companion article (40), reports the effects of O2 manipulation on the field excitatory postsynaptic potential and the antidromic population spike and provides evidence for our hypothesis that the excitatory effects of hyperoxia on the oPS occur primarily by postsynaptic, intrinsic mechanisms of plasticity, whereas the stimulatory effects of hyperoxia to produce sPS (hyperexcitability) occur through disinhibition of spontaneous neurotransmission (40)