Optogenetic methods to record cellular activity

The first genetically encoded calcium indicator (GECI) to be used to image activity in an animal was cameleon, designed by Atsushi Miyawaki, Roger Tsien and coworkers in 1997.[1] Cameleon was first used successfully in an animal by Rex Kerr, William Schafer and coworkers to record from neurons and muscle cells of the nematode C. elegans.[2] Cameleon was subsequently used to record neural activity in flies[3] and zebrafish.[4] In mammals, the first GECI to be used in vivo was GCaMP,[5] first developed by Nakai and coworkers in 2001.[6] GCaMP has undergone numerous improvements, notably by a team of scientists at the Janelia Farm Research Campus (GENIE project, HHMI), and GCaMP6[7] in particular has become widely used in neuroscience. Very recently, G protein-coupled receptors have been harnessed to generate a series of highly specific indicators for various neurotransmitters.[8]

• can be targeted to specific classes of cells (e.g. astrocytes or pyramidal cells). This allows for optical read-out without spatial resolution, e.g. fiber photometry from deep brain areas.[9]
• can be targeted to sub-cellular compartments (e.g. synapses, organelles, nucleus) by fusing the indicator protein with specific anchoring domains, retention signals or intrabodies.
• work in a variety of species (nematodes, insects, fish, mammals) and in cell culture systems (FLIPR assay)
• can be delivered by viral vectors (e.g. rAAV)
• can be used to record the activity of thousands of neurons at the same time [10]

• will buffer the measured ion or protein, potentially interfering with cellular signaling
• are subject to photobleaching, compromising long-term measurements
• can be toxic when expressed at very high concentration
• require highly sensitive cameras or laser scanning microscopes
• most indicators are green fluorescent, making it difficult to measure several cellular parameters simultaneously (multiplexing).

The calcium indicator GCaMP in its calcium-bound (top) and calcium-free form (bottom). When Ca-calmodulin (cyan) binds to M13, the conformation changes and the cpGFP barrel closes, enabling green fluorescence.

Indicators have been designed to measure ion concentrations, membrane potential, neurotransmitters, and various intracellular signaling molecules. The following list provides only examples for each class; many more have been published.

• Genetically encoded calcium indicators (GECI): A large class of tools, based on natural calcium binding proteins (calmodulin, troponin). Different affinities, kinetics, and colors (green, red) available. Read-out via fluorescence intensity (single wavelength indicators), FRET or BRET. Have been targeted to various organelles. Current version: JGCaMP8[11]
• Genetically encoded chloride indicators: Clomeleon[12]
• Genetically encoded indicators for intracellular pH (GEPhI): CypHer[13]
• Genetically encoded voltage indicators (GEVI): ArcLight[14]
• Genetically encoded vesicle fusion sensors: Synapto-pHluorin, Synaptophysin-pHluorin[15]
• Genetically encoded glutamate sensors: GluSnFR[16]
• Genetically encoded GABA sensors: iGABASnFR[17]
• Genetically encoded dopamine sensors: dLight1,[18] GRAB-DA[19]
• Genetically encoded serotonin sensors: GRAB_5-HT[20]
• Genetically encoded norepinephrine sensors: GRAB_NE[21]
• Genetically encoded ATP sensors: QUEEN-37C[22]
• Genetically encoded cAMP sensors: EPAC[23]
• Genetically encoded kinase activity sensors: CaMui,[24] SmURFP
• Genetically encoded Small G-protein sensors: FRas[25]
• Genetically encoded sensor for endocannabinoid activity: GRAB_eCB2.0[26]