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Genetically encoded optical indicators for the analysis of neuronal circuits

Key Points

  • One of the central problems in neuroscience is understanding how behaviour emerges from the cooperative activities of large number of neurons. The workings of neuronal circuits provide a link between behaviour and cellular level signals.

  • Recent research strategies that place neuronal circuits at the centre are progressing rapidly as a result of technological advances that combine genetic manipulation with light-based methods.

  • Among the core tools of these new approaches are genetically encoded optical indicators that enable non-destructive and long-term interrogation of neuronal circuits. Genetic targeting enables the examination of neuronal activities, at either the multiple single-cell or the population level, in defined cell classes.

  • The neuronal activity can be monitored either with genetically encoded calcium indicators (GECIs) or genetically encoded voltage indicators (GEVIs). A great advantage of these tools over low-molecular-mass organic indicators is their permanent labelling and, therefore, the facilitation of long-term (chronic) studies.

  • GECIs and GEVIs have different advantages and limitations. GECIs typically provide a larger signal-to-noise ratio, whereas GEVIs offer better temporal resolution and report subthreshold synaptic information.

  • Use of genetically encoded optical indicators involves appropriate gene targeting and optical imaging methods.

  • In the past few years, genetically encoded optical indicators have been successfully applied to analyse defined cell classes at the population level, to study the anatomical organization of sensory and motor representations, and the dynamics of neuronal circuits. These successes indicate that these methodologies will play an increasingly important role in circuit-centric approaches in neuroscience.

Abstract

In a departure from previous top-down or bottom-up strategies used to understand neuronal circuits, many forward-looking research programs now place the circuit itself at their centre. This has led to an emphasis on the dissection and elucidation of neuronal circuit elements and mechanisms, and on studies that ask how these circuits generate behavioural outputs. This movement towards circuit-centric strategies is progressing rapidly as a result of technological advances that combine genetic manipulation with light-based methods. The core tools of these new approaches are genetically encoded optical indicators and actuators that enable non-destructive interrogation and manipulation of neuronal circuits in behaving animals with cellular-level precision. This Review examines genetically encoded reporters of neuronal function and assesses their value for circuit-oriented neuroscientific investigations.

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Figure 1: Genetically encoded optical indicators of neuronal activity.
Figure 2: Delivery of indicator genes to specified cell types in live animals.
Figure 3: Population responses from genetically targeted cell classes.
Figure 4: Mapping representations onto anatomical space.
Figure 5: Neuronal circuit dynamics.
Figure 6: Chronic imaging in vivo using GECIs.

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Acknowledgements

I thank all members of my laboratory for the data presented in the figures in this article, and for their dedication to the ideas reviewed in this article over the past 15 years. Work in my laboratory is supported by intramural grants from RIKEN; the Japanese Society for Promotion of Science; the Human Frontiers Science Program; the US National Institutes of Health; and the Ministry of Education, Culture, Sport, Science and Technology of Japan.

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Glossary

Systems of circuits

These result from the functional integration of local circuits. For example, sensory–motor integration involves the system of sensory and motor circuitries.

Patch-clamp techniques

These electrophysiological methods were originally developed to resolve the flow of current through a single ion channel in a very small patch of cell membrane in contact with the tip of a fine-glass pipette (such as 1 μm in diameter). These days, the patch-clamp technique is more widely used in a whole-cell configuration with electrical access to the inside of the cell, thereby allowing high-quality microelectrode recordings of membrane voltage.

Dual whole-cell recordings

The use of two patch-clamp microelectrodes to obtain whole-cell recordings from two cells.

Multi-electrode arrays

Devices that provide electrical contact with the extracellular space at many (tens or hundreds) independent points. At each point, electrical signals can be recorded from local single neurons or from cells in the neighbourhood (population signals).

Two-photon (2P) calcium imaging

Recordings of changes in intracellular calcium concentration using fluorescent calcium indicators and 2P excitation microscopy. Allows imaging of many neurons simultaneously in live animals up to about 1mm below the brain surface with micrometre spatial resolution.

Electroencephalography

(EEG). A technique used to measure neural activity by monitoring electrical signals from the brain that reach the scalp. EEG has good temporal resolution but relatively poor spatial resolution.

Magnetoencephelography

(MEG). A method of measuring physiological activity across the cortex by detecting pertubations in the magnetic field that is generated by the electrical activity of neuronal populations.

Local field potential

(LFP). Neuronal signals recorded from the extracellular space, which reflect extracellular currents associated with synaptic potentials and (to a lesser extent) action potentials.

Voltage-sensitive dye imaging

Recordings of membrane voltage transients using voltage-sensitive dyes. Typically, fluorescent dyes and specialized digital cameras are used. When used in brains in live animals, each picture element (pixel) represents the average membrane voltage transients over membranes from many cells.

Cell assemblies

Groups of neurons that perform a given action or represent a given percept or concept in the brain. It has been proposed that anatomically distributed neurons dynamically form assemblies by virtue of transient synchrony.

Förster resonance energy transfer

(FRET). The non-radiative transfer of energy from a donor chromophore, initially in its electronic excited state, to an acceptor chromophore. The efficacy (probability) of transfer depends strongly on the proximity and orientation of the two chromophores. In FRET-based indicators, modulation of FRET efficacy serves as a readout of the structural state of a sensor protein.

Signal-to-noise ratio

(SNR). The ratio between signal size and measurement noise. For optical signals, the maximal achievable SNR is proportional to the square root of number of sampled photons.

Cre recombinase

The enzyme of the P1 bacteriophage that catalyses recombination between two specific short DNA sequences (loxP sites), leading to excision or inversion of the intervening sequence. Genes that are artificially flanked with loxP sites are said to be floxed. Recombination occurs if the cells both carry the floxed genes and express Cre recombinase.

Central pattern generator

A neural circuit that produces self-sustaining (rhythmic) patterns of neuronal activity and behaviour without requiring sensory feedback.

Glomeruli

Glomeruli comprise specialized structures in the olfactory bulb and consist of incoming olfactory sensory neuron axons, the dendrites of both second-order projection neurons and local interneurons, and the processes of astrocytes.

Place cells

Neurons in the hippocampus and parahippocampus that show increased frequency of firing when an animal is in a specific area referred to as the cell's place field.

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Knöpfel, T. Genetically encoded optical indicators for the analysis of neuronal circuits. Nat Rev Neurosci 13, 687–700 (2012). https://doi.org/10.1038/nrn3293

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