support-information-section).

Objective A fundamental challenge in optogenetics is to elicit long-term high-fidelity neuronal spiking with negligible heating. Fast channelrhodopsins (ChRs) require higher irradiances and cause spike failure due to photocurrent desensitization under sustained illumination, whereas, more light-sensitive step-function opsins (SFOs) exhibit prolonged depolarization with insufficient photocurrent and fast response for high-fidelity spiking. Approach We present a novel method to overcome this fundamental limitation by co-expressing fast ChRs with SFOs. A detailed theoretical analysis of ChETA co-expressed with different SFOs, namely ChR2(C128A), ChR2(C128S), SSFO and SOUL, expressing hippocampal neurons has been carried out by formulating their accurate theoretical models. Main results ChETA-SFO-expressing hippocampal neurons show a more stable photocurrent that overcomes spike failure. Spiking fidelity in these neurons can be sustained even at lower irradiances of subsequent pulses (77 % of initial pulse intensity in ChETA-ChR2(C128A)-expressing neurons) or by using red-shifted light pulses at appropriate intervals. High-fidelity spiking up to 60 Hz can be evoked in ChR2-C128S-ChETA-expressing neurons, which cannot be attained with only SFOs. Significance The present study provides important insights about photostimulation protocols for bi-stable switching of neurons. This new approach provides a means for sustained low-power, high-frequency, and high-fidelity optogenetic switching of neurons, necessary to study various neural functions and neurodegenerative disorders and enhance the utility of optogenetics for biomedical applications.

The fundamental process of information processing and memory formation in the brain is associated with complex neuron firing patterns, which can occur spontaneously or be triggered by sensory inputs. Optogenetics has revolutionized neuroscience by enabling precise manipulation of neuronal activity patterns in specified neural populations using light. However, the light pulses used in optogenetics have been primarily restricted to square waveforms. Here, we present a detailed theoretical analysis of the temporal shaping of light pulses in optogenetic excitation of hippocampal neurons and neocortical fast-spiking interneurons expressed with ultrafast (Chronos), fast (ChR2), and slow (ChRmine) channelrhodopsins. Optogenetic excitation has been studied with light pulses of different temporal shapes that include square, forward-/backward ramps, triangular, left-/right-triangular, Gaussian, left-/right-Gaussian, positive-sinusoidal, and left-/right-positive sinusoidal. Different light shapes result in significantly different photocurrent amplitudes and kinetics, spike-timing, and spontaneous firing rate. For short duration stimulations, left-Gaussian pulse results in larger photocurrent in ChR2 and Chronos than square pulse of the same energy density. Time to peak photocurrent in each opsin is minimum at right-Gaussian pulse. The optimal pulse width to achieve peak photocurrent for non-square pulses is 10 ms for Chronos, and 50 ms for ChR2 and ChRmine. The pulse energy to evoke spike in hippocampal neurons can be minimized on choosing square pulse with Chronos, Gaussian pulse with ChR2, and positive-sinusoidal pulse with ChRmine. The results demonstrate that non-square waveforms generate more naturalistic spiking patterns compared to traditional square pulses. These findings provide valuable insights for the development of new optogenetic strategies to better simulate and manipulate neural activity patterns in the brain, with the potential to improve our understanding of cognitive processes and the treatment of neurological disorders.

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