Rationale:
Virtually all seizures terminate naturally, without medical intervention. Seizure termination often coincides with instances of sustained neuronal depolarization and suppression of firing, termed cortical spreading depression (CSD). We recently showed that CSDs can be induced by halorhodopsin activation (Parrish et al, PMID 36639898), leading us to hypothesize that the specific initiation step is an osmotic crisis, arising from the redistribution of ions into neurons (Ricks et al, PMID 40448933). To explore this mechanism further, we have developed a new method of inducing transient intense depolarisation events, in single neurons, allowing pharmacological investigations of the phenomenon.
Methods:
Brain slices are prepared acutely from young adult wild-type mice. These are bathed in conventional artificial CSF solution, and under DIC visualization, we made recordings of pyramidal cells in whole cell patch clamp mode, using a K-gluconate electrode filling solution with approximately 15mM intracellular Cl-.
Results:
We report that when neurons are held persistently at a hyperpolarized level (usually -100mV), simulating the effect of halorhodopsin activation, they are typically stable for a period of time, before showing a sudden, very large increase in holding current, indicative of the opening of a large depolarizing conductance. At this time, cells become permeable to sulforhodamine, a large organic fluorescent molecule. If the holding potential is then moved to -70mV, cells recover progressively over the subsequent 100s, and the input resistance returns to baseline levels. These transient depolarizing events are accompanied by a large rise in intracellular Ca2+. The reversal potential of these transient conductances was very positive, and was further positive-shifted when using KCl-based electrode-filling solution, indicating a large Cl—component to the conductance. We recorded such events in over 60% of recordings, but the proportion of neurons was markedly lower when voltage-regulated anion channels (VRAC) and transient-response potential (TRP)-M7 channels were blocked pharmacologically.Conclusions:
We demonstrate the occurrence of very large, transient conductances, that can be induced in cortical neurons by sustained hyperpolarization. Pharmacological analyses of this conductance are consistent with those seen in non-neuronal cells triggered by osmotic tension.
Funding: Epilepsy Research Institute, UK, project P2502