Long-term in vivo application of a potassium channel-based optogenetic silencer in the healthy and epileptic mouse hippocampus

Abstract

Background Optogenetic tools allow precise manipulation of neuronal activity via genetically encoded light-sensitive proteins. Currently available optogenetic inhibitors are not suitable for prolonged use due to short-lasting photocurrents, tissue heating, and unintended changes in ion distributions, which may interfere with normal neuron physiology. To overcome these limitations, a novel potassium channel-based optogenetic silencer, named PACK, was recently developed. The PACK tool has two components: a photoactivated adenylyl cyclase from Beggiatoa (bPAC) and a cAMP-dependent potassium channel, SthK, which carries a large, long-lasting potassium current in mammalian cells. Previously, it has been shown that activating the PACK silencer with short light pulses led to a significant reduction of neuronal firing in various in vitro and acute in vivo settings. Here, we examined the viability of performing long-term studies in vivo by looking at the inhibitory action and side effects of PACK and its components in healthy and epileptic adult male mice. Results We targeted hippocampal cornu ammonis (CA1) pyramidal cells using a viral vector and enabled illumination of these neurons via an implanted optic fiber. Local field potential (LFP) recordings from CA1 of freely moving mice revealed significantly reduced neuronal activity during 50-min intermittent (0.1 Hz) illumination, especially in the gamma frequency range. Adversely, PACK expression in healthy mice induced chronic astrogliosis, dispersion of pyramidal cells, and generalized seizures. These side effects were independent of the light application and were also present in mice expressing bPAC without the potassium channel. Light activation of bPAC alone increased neuronal activity, presumably via enhanced cAMP signaling. Furthermore, we applied bPAC and PACK in the contralateral hippocampus of chronically epileptic mice following a unilateral injection of intrahippocampal kainate. Unexpectedly, the expression of bPAC in the contralateral CA1 area was sufficient to prevent the spread of spontaneous epileptiform activity from the seizure focus to the contralateral hippocampus. Conclusion Our study highlights the PACK tool as a potent optogenetic inhibitor in vivo. However, further refinement of its light-sensitive domain is required to avoid unexpected physiological changes.

Rare KCND3 Loss-of-Function Mutation Associated With the SCA19/22

“…Among the ion channels, potassium channels have the most subtypes and the most complex functions and are the focus of much clinical and scientific research. For example, research has been conducted on voltage-gated potassium channels, such as Kv1, Kv2, Kv3, and Kv4 (Heijman et al, 2021 ; Ikeno et al, 2021 ; Kleis et al, 2022 ). The Kv family of potassium channels are glycosylated polypeptide complexes composed of four α-subunits and four β-auxiliaries.…”

Aion is a bistable anion-conducting channelrhodopsin that provides temporally extended and reversible neuronal silencing

“…While providing a promising new direction for efficient, light-sensitive silencing, putative caveats are off-target effects due to light-induced elevation of cyclic nucleotides and background-activation of the cyclic nucleotide-gated K + -channels by endogenous cyclic nucleotides. Such side effects may explain the unexpected alterations of hippocampal activity in mice expressing these tools 43 . The recently discovered natural K + channels 44 might provide a solution to such limitations.…”

Neurophotonic methods in approach to in vivo animal epileptic models: Advantages and limitations

“…To a large extent, these shortcomings are avoided in a new optogenetic potassium channel silencer called PACK. 96 The PACK tool consists of two components: Beggiatoa's photoactivated adenylate cyclase (bPAC) and the cyclic adenosine monophosphatedependent potassium channel SthK. This channel provides a continuous potassium current.…”

“…Activation of the PACK silencer by short light pulses causes a decrease in neuronal excitation both in vitro and in vivo. 96 A study of the consequences of ictal events, namely, sudden unexpected death in epilepsy (SUDEP), has also been attempted using optogenetic methods. 97 Seizureinduced respiratory arrest (S-IRA) has been described both in patients and in animal models.…”