Neuronal hyperactivity causes Na⁺/H⁺ exchanger-induced extracellular acidification at active synapses

Extracellular pH influences neuronal activity, and neuronal activity in turn affects extracellular H+ concentration. Prior research has shown that pH fluctuations occur in response to neural activity. However, the specific dynamics of pH changes at synaptic sites during hyperexcitability remain unclear. This gap motivated the development of a new pH indicator to monitor extracellular pH in real time. No prior work had resolved the spatial and temporal patterns of pH shifts during seizures or hyperactivity. The need to distinguish between various acidification mechanisms was unmet. Existing studies lacked the tools to track pH changes at active synapses. This study aimed to clarify the source of extracellular acidification during neuronal hyperactivity. Understanding these dynamics could improve models of synaptic pH regulation.

The study aimed to investigate how extracellular pH changes at synaptic sites during neuronal hyperexcitability. Specifically, the researchers wanted to determine the spatial and temporal patterns of pH fluctuations. They also sought to identify the mechanism responsible for extracellular acidification. The motivation stemmed from the lack of real-time pH monitoring tools in synaptic regions. Previous work could not distinguish between vesicle release and transporter activity as causes of pH shifts. The researchers proposed using a novel pH indicator to track these changes. Their goal was to link synaptic activity to pH dynamics in a controlled setting. This could help clarify the role of pH in seizure-related brain activity.

The researchers developed ex.E2GFP, a membrane-targeted extracellular pH indicator. This tool was engineered to detect acidic shifts with high sensitivity. They used primary cortical neurons cultured in vitro for their experiments. Neuronal hyperexcitability was induced using convulsant drugs or high-frequency stimulation. Real-time fluorescence measurements tracked pH changes in the extracellular space. The location of pH shifts was monitored at active synapses. The team tested whether vesicle H+ release or H+ V-ATPase activity caused the shifts. They ruled out these mechanisms and focused on the role of the Na+/H+ exchanger.

Neuronal hyperactivity led to extracellular pH decreases at active synapses. This acidic shift was reversible when activity was silenced. The pH change occurred specifically at synapses involved in active signaling. Vesicle H+ outflow did not account for the observed acidification. Membrane-exposed H+ V-ATPase activity was also not responsible for the shift. Instead, the Na+/H+ exchanger was identified as the main contributor. The pH decrease was localized and transient, returning to baseline after activity ceased. These findings suggest a direct link between synaptic activity and extracellular pH regulation.

The study demonstrates that extracellular pH shifts occur during neuronal hyperexcitability. These changes are localized to active synapses and reversible. The Na+/H+ exchanger is the primary mechanism behind the acidification. Vesicle release and H+ V-ATPase activity were not responsible for the observed pH changes. The findings emphasize the tight coupling between synaptic activity and pH dynamics. This evidence supports the idea that pH regulation is a key factor in neural hyperactivity. The results may help explain the physio-pathological mechanisms in epileptic brains. The study contributes to understanding how pH fluctuations influence seizure activity.