Overexpression of KCNN4 channels in principal neurons produces an anti-seizure effect without reducing their coding ability.

Gene therapy offers a potential alternative to the surgical treatment of epilepsy, which affects millions of people and is pharmacoresistant in ~30% of cases. Aimed at reducing the excitability of principal neurons, the engineered expression of K+ channels has been proposed as a treatment due to the outstanding ability of K+ channels to hyperpolarize neurons. However, the effects of K+ channel overexpression on cell physiology remain to be investigated. Here we report an adeno-associated virus (AAV) vector designed to reduce epileptiform activity specifically in excitatory pyramidal neurons by expressing the human Ca2+-gated K+ channel KCNN4 (KCa3.1). Electrophysiological and pharmacological experiments in acute brain slices showed that KCNN4-transduced cells exhibited a Ca2+-dependent slow afterhyperpolarization that significantly decreased the ability of KCNN4-positive neurons to generate high-frequency spike trains without affecting their lower-frequency coding ability and action potential shapes. Antiepileptic activity tests showed potent suppression of pharmacologically induced seizures in vitro at both single cell and local field potential levels with decreased spiking during ictal discharges. Taken together, our findings strongly suggest that the AAV-based expression of the KCNN4 channel in excitatory neurons is a promising therapeutic intervention as gene therapy for epilepsy.

Optogenetic Low-Frequency Stimulation of Principal Neurons, but Not Parvalbumin-Positive Interneurons, Prevents Generation of Ictal Discharges in Rodent Entorhinal Cortex in an In Vitro 4-Aminopyridine Model.

Low-frequency electrical stimulation is used to treat some drug-resistant forms of epilepsy. Despite the effectiveness of the method in suppressing seizures, there is a considerable risk of side effects. An optogenetic approach allows the targeting of specific populations of neurons, which can increase the effectiveness and safety of low-frequency stimulation. In our study, we tested the efficacy of the suppression of ictal activity in entorhinal cortex slices in a 4-aminopyridine model with three variants of low-frequency light stimulation (LFLS): (1) activation of excitatory and inhibitory neurons (on Thy1-ChR2-YFP mice), (2) activation of inhibitory interneurons only (on PV-Cre mice after virus injection with channelrhodopsin2 gene), and (3) hyperpolarization of excitatory neurons (on Wistar rats after virus injection with archaerhodopsin gene). Only in the first variant did simultaneous LFLS of excitatory and inhibitory neurons replace ictal activity with interictal activity. We suggest that LFLS caused changes in the concentration gradients of K+ and Na+ cations across the neuron membrane, which activated Na-K pumping. According to the mathematical modeling, the increase in Na-K pump activity in neurons induced by LFLS led to an antiepileptic effect. Thus, a less specific and generalized optogenetic effect on entorhinal cortex neurons was more effective in suppressing ictal activity in the 4-aminopyridine model.

Photostimulation activates fast-spiking interneurons and pyramidal cells in the entorhinal cortex of Thy1-ChR2-YFP line 18 mice.

The application of optogenetics in animals has provided new insights into both fundamental neuroscience and diseases of the nervous system. This is primarily due to the fact that optogenetics allows selectively activating or inhibiting particular types of neurons. One of the first transgenic mouse lines developed for the optogenetic experiment was Thy1-ChR2-YFP. Thy1 is an immunoglobulin superfamily member expressing in projection neurons, so it was assumed that channelrhodopsin-2 (ChR2) would be primarily expressed in projection neurons. However, the specificity of ChR2 expression under promoter Thy1 in different lines has to be clarified yet. Therefore, we aimed to determine the cell specificity of ChR2 expression in the entorhinal cortex of Thy1-ChR2-YFP line 18 mice. We have found that both pyramidal cells and fast-spiking interneurons in deep layers of the entorhinal cortex depolarized and fired in response to 470-nm photostimulation. To exclude the effect of synaptic activation of interneurons by pyramidal cells, we used a selective antagonist of AMPA receptors. Under these conditions, inhibitory postsynaptic currents decreased but did not disappear completely. Furthermore, gabazine inhibited these postsynaptic currents entirely, thus confirming the direct activation of interneurons by light. These data demonstrate that ChR2 is expressed in both pyramidal neurons and fast-spiking interneurons of the entorhinal cortex in Thy1-ChR2-YFP mice.