Second-generation optogenetic control of neurons: bistable and near-infrared-activated channels for in Vivo applications

The first decade of optogenetics, anchored on channelrhodopsin-2 and its early variants, established the basic capability of millisecond-precision genetic control over identified neurons but exposed three persistent technical constraints: shallow tissue penetration of blue light, the requirement for high light intensities that produce phototoxicity in chronic protocols, and the obligatory presence of an implanted intracranial fibre. Between 2016 and 2022, a second generation of optogenetic actuators substantially relaxed each of these constraints. Ultra-sensitive bistable step-function opsins (SSFO derivatives, SOUL) lowered the photon flux required for action potential generation by approximately three orders of magnitude and extended the depolarisation lifetime to tens of minutes. Red-shifted and near-infrared (NIR) activated channels (ReaChR derivatives, ChrimsonR, ChRmine and its engineered variants) shifted the action spectrum into the optical tissue window where blood and water attenuation are minimised. Upconversion-nanoparticle (UCNP) hybrid systems coupled tissue-penetrating NIR illumination to visible-light-responsive opsins through molecularly tailored lanthanide-doped nanocrystals. Combined with soma-targeting motifs (ST-ChroME, somBiPOLES) and two-photon holographic illumination, these tools enabled, by 2022, transcranial deep-brain stimulation of identified circuits at depths exceeding 7 mm in mice and single-cell-resolution simultaneous control of dozens of neurons in cortex. The accelerating pace of new tool publications has, however, made comparative tool selection for a specific in vivo application increasingly difficult. In this article I review the technical landscape of second-generation actuators along five performance dimensions — light sensitivity, kinetics, spectral red-shift, soma-specificity, and depth-reachability — and propose the In Vivo Optogenetic Tool Selection Index (IOTSI), a single normalised composite metric — bounded on [0,1] — that integrates the five dimensions and predicts the optimal actuator class for a defined experimental design. IOTSI applied to ten representative second-generation tools returns a tool-by-application ranking that, on the basis of currently published in vivo data, prefers ChRmine-class actuators for transcranial deep-brain stimulation, SOUL-class step-function opsins for chronic minimally-invasive protocols, and STChroME or somBiPOLES for two-photon holographic single-cell control