Optogenetics is neural stimulation by light: a delivered gene makes chosen neurons express a light-gated ion channel, and illumination opens it. In mammalian neurons, switched light controlled spiking with millisecond timing; in living mouse cortex, holographic multiphoton illumination targeted single neurons in three dimensions, exciting pyramidal cells or silencing identified neurons through an inhibitory anion channel. What it reads is a check on the result: electrical recordings of spiking, which are direct, or calcium fluorescence, which is indirect.
Spatial resolution
11 µm (suprathreshold extent)
Temporal resolution
3 ms (inter-pulse interval)
Field of view
30250000 µm³ (reachable envelope)
Neurons covered
346 neurons (suprathreshold volume)
Reads
photocurrents by electrophysiology; evoked or suppressed spiking by electrophysiology; GCaMP6s calcium fluorescence by imaging
Writes
High-speed switched light delivered to ChR2-expressing mammalian neurons; 3D-SHOT multiphoton illumination delivered to in vivo cortical neurons expressing soma-targeted ChroME or IRES-ST-eGtACR1
Destructive?
minimally destructive
Prerequisites
genetic engineering: Inject AAV encoding the opsin and, depending on the combination, Cre or GCaMP6. Use mice 2–6 weeks after opsin injection or 1–2 weeks after GCaMP6 injection.; germline genetic engineering: For specified combinations, use inherited CamKII-tTA × tetO-GCaMP6s, CamKII-tTA × tetO-GCaMP6s × Emx1-Cre, or PV-Cre mice. These lines are protocol-specific and distinct from somatic viral expression.; anesthesia: For viral injection, anesthetize mice with 2% isoflurane. For headplate and window surgery, use 2% isoflurane, 2 mg/kg dexamethasone and 0.05 mg/kg buprenorphine (written 'bupromorphine' in the protocol).; surgery: Fit a stainless-steel headplate, make a 3 mm craniotomy over left primary somatosensory cortex and permanently seal it with a coverslip window.; other: Allow heated recovery. Starting two days after surgery, habituate mice to head fixation over a freely moving circular treadmill for 2–7 days; subsequent calcium imaging is head-fixed on a freely spinning wheel.
Theoretical field of view
206910000 µm³ (reachable envelope)
Description
Optogenetic neural stimulation couples genetically expressed light-sensitive proteins to controlled illumination. Lentiviral delivery of a rapidly gated, light-sensitive cation channel, combined with high-speed optical switching, provides millisecond control of spiking and excitatory and inhibitory synaptic transmission in mammalian neurons. In living cortex, soma-targeted excitatory opsins activate pyramidal neurons, while a soma-targeted inhibitory anion opsin suppresses activity in identified neurons. Multiphoton holographic illumination with temporal focusing targets cortical neurons across three dimensions, with cellular spatial resolution, patterned ensembles and ordered stimulation sequences. Volumetric two-photon calcium imaging provides an optical activity readout alongside stimulation. Electrophysiological recordings also demonstrate silencing.