Carbogen-Induced Respiratory Acidosis Blocks Experimental Seizures by a Direct and Specific Inhibition of Na_V1.2 Channels in the Axon Initial Segment of Pyramidal Neurons

Abstract

Brain pH is a critical factor for determining neuronal activity, with alkalosis increasing and acidosis reducing excitability. Acid shifts in brain pH through the breathing of carbogen (5% CO₂/95% O₂) reduces seizure susceptibility in animal models and patients. The molecular mechanisms underlying this seizure protection remain to be fully elucidated. Here, we demonstrate that male and female mice exposed to carbogen are fully protected from thermogenic-triggered seizures. Whole-cell patch-clamp recordings revealed that acid shifts in extracellular pH (pHo) significantly reduce action potential firing in CA1 pyramidal neurons but did not alter firing in hippocampal inhibitory interneurons. In real-time dynamic clamp experiments, acidification reduced simulated action potential firing generated in hybrid model neurons expressing the excitatory neuron predominant Na_V1.2 channel. Conversely, acidification had no effect on action potential firing in hybrid model neurons expressing the interneuron predominant Na_V1.1 channel. Furthermore, knockdown of Scn2a mRNA in vivo using antisense oligonucleotides reduced the protective effects of carbogen on seizure susceptibility. Both carbogen-mediated seizure protection and the reduction in CA1 pyramidal neuron action potential firing by low pHo were maintained in an Asic1a knock-out mouse ruling out this acid-sensing channel as the underlying molecular target. These data indicate that the acid-mediated reduction in excitatory neuron firing is mediated, at least in part, through the inhibition of Na_V1.2 channels, whereas inhibitory neuron firing is unaffected. This reduction in pyramidal neuron excitability is the likely basis of seizure suppression caused by carbogen-mediated acidification.

SIGNIFICANCE STATEMENT Brain pH has long been known to modulate neuronal excitability. Here, we confirm that brain acidification reduces seizure susceptibility in a mouse model of thermogenic seizures. Extracellular acidification reduced excitatory pyramidal neuron firing while having no effect on interneuron firing. Acidification also reduced dynamic clamp firing in cells expressing the Na_V1.2 channel but not in cells expressing Na_V1.1 channels. In vivo knockdown of Scn2a mRNA reduced seizure protection of acidification. In contrast, acid-mediated seizure protection was maintained in the Asic1a knock-out mouse. These data suggest Na_V1.2 channel as an important target for acid-mediated seizure protection. Our results have implications on how natural variations in pH can modulate neuronal excitability and highlight potential antiseizure drug development strategies based on the Na_V1.2 channel.