Optimized photo-stimulation of halorhodopsin for long-term neuronal inhibition.

BACKGROUND: Optogenetic silencing techniques have expanded the causal understanding of the functions of diverse neuronal cell types in both the healthy and diseased brain. A widely used inhibitory optogenetic actuator is eNpHR3.0, an improved version of the light-driven chloride pump halorhodopsin derived from Natronomonas pharaonis. A major drawback of eNpHR3.0 is related to its pronounced inactivation on a time-scale of seconds, which renders it unsuited for applications that require long-lasting silencing. RESULTS: Using transgenic mice and Xenopus laevis oocytes expressing an eNpHR3.0-EYFP fusion protein, we here report optimized photo-stimulation techniques that profoundly increase the stability of eNpHR3.0-mediated currents during long-term photo-stimulation. We demonstrate that optimized photo-stimulation enables prolonged hyperpolarization and suppression of action potential discharge on a time-scale of minutes. CONCLUSIONS: Collectively, our findings extend the utility of eNpHR3.0 to the long-lasting inhibition of excitable cells, thus facilitating the optogenetic dissection of neural circuits.

Altered Chloride Homeostasis Decreases the Action Potential Threshold and Increases Hyperexcitability in Hippocampal Neurons.

Chloride ions play an important role in controlling excitability of principal neurons in the central nervous system. When neurotransmitter GABA is released from inhibitory interneurons, activated GABA type A (GABAA) receptors on principal neurons become permeable to chloride. Typically, chloride flows through activated GABAA receptors into the neurons causing hyperpolarization or shunting inhibition, and in turn inhibits action potential (AP) generation. However, in situations when intracellular chloride concentration is increased, chloride ions can flow in opposite direction, depolarize neurons, and promote AP generation. It is generally recognized that altered chloride homeostasis per se has no effect on the AP threshold. Here, we demonstrate that chloride overload of mouse principal CA3 pyramidal neurons not only makes these cells more excitable through GABAA receptor activation but also lowers the AP threshold, further aggravating excitability. This phenomenon has not been described in principal neurons and adds to our understanding of mechanisms regulating neuronal and network excitability, particularly in developing brain and during pathological situations with altered chloride homeostasis. This finding further broadens the spectrum of neuronal plasticity regulated by ionic compositions across the cellular membrane.