Understanding the intricate connections between neurons and their roles in driving behavior is a central challenge in neuroscience. Current methods for studying neural activity, such as patch-clamp electrophysiology, are invasive and have limited throughput. Calcium imaging, while applicable in vivo, suffers from low temporal resolution and cannot resolve subthreshold voltage transients or high-frequency action potentials. Genetically encoded voltage indicators (GEVIs) offer improved temporal resolution but are often used with static optogenetic stimulation, leading to adaptation and incomplete control of neural activity. This study aimed to develop an all-optical voltage-clamp (OVC) system that combines the non-invasive nature of optical methods with the precise control offered by electrophysiology. Such a system would provide a powerful tool for studying neural circuits and performing high-throughput screening in excitable cells. Previous attempts at optical control, such as (i)-Optopatch and optoclamp, either lacked bidirectional control or relied on indirect voltage readouts, limiting their precision. This research sought to address these limitations by developing a closed-loop system that uses a GEVIs for voltage read-out and a tandem protein actuator, BiPOLES, capable of both depolarization and hyperpolarization, to achieve true all-optical voltage clamping.

The paper reviews existing methods for controlling and observing excitable cell function, highlighting the limitations of invasive patch-clamp electrophysiology and the lower temporal resolution of calcium imaging. It discusses the advantages of GEVIs, particularly rhodopsin-based indicators, for monitoring voltage dynamics. The authors cite previous work on unidirectional optical steering of neuronal activity using ChR and QuasAr2, as well as the use of archaerhodopsin in optical dynamic clamp experiments and bidirectional optical modulation in light-induced electrophysiology (LiEp). The need for a true all-optical voltage-clamp with closed-loop feedback is emphasized, emphasizing the shortcomings of existing approaches that either lacked bidirectional control or used indirect voltage readouts.

The researchers developed an optogenetic voltage-clamp (OVC) system using the voltage indicator QuasAr2 and the bidirectional optogenetic actuator BiPOLES. BiPOLES, a tandem protein consisting of Chrimson (depolarizer) and GtACR2 (hyperpolarizer), allows for precise bidirectional control of membrane potential. QuasAr2 fluorescence, excited by a 637 nm laser, was used to monitor membrane voltage. A custom-written Beanshell script processed the fluorescence signals, comparing them to target values and adjusting the wavelength of light delivered to BiPOLES via a monochromator to maintain the desired voltage. The system's performance was characterized in *C. elegans* body-wall muscle cells (BWMs) and cholinergic and GABAergic motor neurons, and subsequently in rat hippocampal neurons in organotypic slice culture. Simultaneous measurements of fluorescence and voltage using patch-clamp electrophysiology allowed calibration of fluorescence signals to actual membrane voltages and wavelengths to currents, enabling the determination of optical I/V relationships. The authors compared the performance of their system using a single actuator (ChR2 or GtACR2) versus BiPOLES and used a decision tree algorithm for feedback control, finding BiPOLES to be significantly faster. To assess homeostatic changes in cellular physiology, they used the OVC on *unc-13* and *egl-19* mutants. They also explored the system's capability for dynamic clamping of spontaneous activity in the *C. elegans* pharynx and DVB neuron. For mammalian neurons, the OVC protocol was adapted, but the voltage range was smaller due to the resting potential being closer to the Cl⁻ reversal potential. Electrophysiological recordings were performed using standard patch-clamp techniques. Statistical analyses included one-way ANOVA, t-tests, and linear regressions.

The OVC successfully clamped membrane voltage in *C. elegans* BWMs, cholinergic and GABAergic neurons, and rat hippocampal neurons. In *C. elegans*, the system achieved a voltage range of approximately 15 mV per 6% ΔF/F0 in BWMs, and a larger range in DVB neurons. The system's speed was significantly improved using BiPOLES compared to single actuators. The OVC detected altered excitability in *unc-13* mutants, indicating its sensitivity to homeostatic changes. Optical I/V relationships were successfully obtained in *C. elegans*, showing good agreement with electrophysiological data. The OVC dynamically clamped spontaneous rhythmic activity in the *C. elegans* pharynx and DVB neuron, suppressing associated behaviors. In mammalian neurons, the OVC was effective, but with a more limited voltage range due to the resting potential being close to the Cl⁻ reversal potential and potential crosstalk between the excitation light and BiPOLES components. In addition, the OVC allows for "on-the-run" control, dynamically altering clamping parameters during an ongoing experiment. Overall, the OVC was shown to be reliable, fast, and versatile across diverse excitable cells, exceeding the capabilities of existing methods.

The successful implementation of the all-optical voltage clamp represents a significant advance in optogenetics. The OVC surpasses previous approaches by combining bidirectional optogenetic actuation with closed-loop feedback based on real-time GEVIs fluorescence readings. This allows for precise control and observation of membrane voltage in diverse cell types, even during dynamic activity patterns. The ability to generate optical I/V relationships and detect homeostatic changes in mutants opens new avenues for studying cellular physiology. The system’s high throughput and non-invasive nature are significant advantages over traditional electrophysiological methods. While the current speed is slower than patch-clamp, the system's sensitivity and accuracy are comparable within experimental variations. Future improvements, such as using faster optogenetic tools and integrated circuitry, could increase sampling rates and further enhance performance. The study's findings pave the way for all-optical control of individual neurons in freely behaving animals and offer significant promise for high-throughput drug screening and the development of adaptive therapeutic interventions.

This research successfully developed and validated a novel all-optical voltage-clamp (OVC) system for precise control of excitable cells in live animals. The OVC's ability to clamp voltage in diverse cell types, detect subtle physiological changes, and dynamically suppress spontaneous activity marks a significant advancement in optogenetics. Future work could focus on improving the system's speed and expanding its application to a wider range of model organisms and cell types. The potential applications of OVC in high-throughput screening, disease modeling, and adaptive neuromodulation are considerable.

The current version of the OVC is limited by its speed, with transition times of around 90-150 ms, although this is significantly faster than previous attempts at similar technologies. This limitation is primarily due to the software's current maximum sampling rate of 100 Hz and could be improved with faster optogenetic tools and faster processing hardware. The voltage range achievable in mammalian neurons was limited by the resting membrane potential and potential crosstalk between the excitation light and BiPOLES components. Furthermore, the OVC requires calibration, adding an initial step to the experimental process.