REVIEWS

NEURAL CIRCUITS

Genetically encoded optical indicators

for the analysis of neuronal circuits
Thomas Knöpfel
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.

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.
RIKEN Brain Science Institute,
2–1 Hirosawa, Wako City,
Saitama 351–0198, Japan.
e‑mail:
tknopfel@knopfel-lab.net
doi:10.1038/nrn3293
Published online
30 August 2012
Understanding how local neuronal circuits and systems
of circuits transform the activities of individual neurons
into complex behaviours such as sensory–motor integra-
tion, perception, memory formation and mental state is
one of the key challenges of contemporary neuroscience.
The term ‘neuronal circuit’ is used in many ways; how-
ever, the simplest anatomy-based definition states that
a circuit consists of two or more interconnected neu-
rons (this number might be as high as 1 × 1011 neurons
if the entire human brain is considered as an integrated
neuronal circuit). Neuronal circuits display emergent
features such as synchrony 1–5, oscillations1,6, waves7 and
sequential activation patterns8–10 that cannot be deduced
by examining the activity of individual neurons. The
basic workings of neuronal circuits can be studied in
isolated brain tissue, but mental processes presumably
emerge only in intact brains. Whether in isolated tissue
or the intact brain, the study of neuronal circuits requires
methods to monitor the activity of large numbers of
neurons simultaneously 11–14.
One widely adopted approach to analyse the inter-
actions of many neurons expands upon the concepts
and methodologies of single-cell electrophysiology.
Patch-clamp techniques in brain slices have been paral-
lelized to enable recordings to be made from several
cells simultaneously 15 and, in what is perhaps an even
more challenging task, dual whole-cell recordings have
been achieved in awake rodents16,17. Even larger num-
bers of cells (~200) can be analysed using multi-electrode
arrays12,18. Two-photon (2P) calcium imaging is a powerful
alternative strategy for recording the activity of hundreds
of individual neurons simultaneously in live animals.
2P calcium imaging reduces the temporal resolution (in
comparison to electrophysiology), but this is counter-
balanced by the precise spatial localization of each of the
large number of cells that are monitored19–21.
The use of these bottom‑up strategies to link patterns
of action potentials to behaviour 12,22 are complemented
by top-down strategies that correlate the complex
behaviour that is generated by circuits across multiple
brain regions with signals that are attributable to neu-
ronal ensembles as a whole4,6,23. These population sig-
nals are traditionally observed with techniques such as
electroencephalography (EEG), magnetoencephelography
(MEG), local field potential (LFP) recordings24 and voltage-
sensitive dye imaging25. These signals correlate not only
with primary sensory inputs, motor outputs and brain
states26,27, but also with complex behaviours including
attention27, decision-making 28 and other cognitive pro-
cesses29–32. Moreover, classical microstimulation33–35 and
more recent optogenetic population stimulation experi-
ments have indicated that activation of whole popula-
tions of neurons can cause or influence behaviour 36–42.
Thus, population-level signals reflect circuit operations
(neuronal computations) that link patterns of individual
action potentials with behaviour 43.
Further progress in deducing circuit operations (neu-
ronal computations) from population signals would
greatly benefit from methods that monitor the activity
of specific classes of cells instead of poorly defined cell

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REVIEWS
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 1 mm 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.
688 | O CTOBER 2012 | VOLUME 13 www.nature.com/reviews/neuro
populations at given tissue coordinates. In this context,
ensemble-level approaches reach the same limitations as
bottom‑up cellular approaches that are based on record-
ings of spike trains from large numbers of primarily
unidentified cells. The methodological and conceptual
gap between these two approaches defines the stage for
circuit-centric strategies that focus on large subpopula-
tions of neurons while fully appreciating the diversity
of cellular functions within the overall population. In
this article, I show that genetic methods that target the
expression of optical activity indicators to specific cell
classes provide an important component of these circuit-
centric approaches. The general principles described are
illustrated using examples that are mainly taken from
studies using mice, but their validity is not limited to
this species.
Optical monitoring of neural circuits
The activity of large numbers of neurons can be moni-
tored simultaneously using large-scale parallelized micro-
electrode recordings or optical methods (BOX 1). In this
Review, I focus on optical recording approaches, which
have the advantage of allowing researchers to zoom in to
the microscopic level and out to the macroscopic level
in a straightforward manner, as is required for a circuit-
centric strategy. However, it is important to point out
that zooming out — that is, increasing the number of
cells simultaneously monitored — typically reduces the
level of detailed information that can be gained about
individual cells. For instance, 2P in vivo calcium imag-
ing can be tuned to determine the exact timing of single
action potentials in smaller numbers (<100) of cells44
than is possible if cells are monitored only for periods of
high-frequency firing (up to 500 cells)19–21.
Imaging at the population level without single-cell
resolution drastically increases the number of cells that
can be recorded from, but such population signals are
typically blind to cellular diversity. The lack of specificity
for a cell class is problematic in view of the fact that neu-
ronal circuits typically involve various neuronal classes,
each of which has a specific function. For this reason,
it is common practice in single-cell electrophysiology
experiments to describe the cellular features of each class
of neurons separately.
Genetically encoded optical indicators can overcome
these limitations by enabling cell class-specific optical
recording of the activities of large numbers of neurons
at both single-cell resolution and ensemble levels13.
The advantage of using genetic methods to associate
electrophysiological recordings with cell class identity
was initially realized with the use of transgenic mice
in which specific cells classes were tagged by fluores-
cent proteins45,46. Genetic targeting of optical indica-
tor expression allows neuronal activity to be recorded
directly from identified (and preselected) cell classes.
Importantly, the identity of the selected cell class can be
defined by their anatomical localization (as is the case
in traditional imaging techniques), as well as by its gene
expression profile, connectivity and the cells with which
they are synchronously recruited during formation
of cell assemblies.
© 2012 Macmillan Publishers Limited. All rights reserved
Methodologies and technologies
Design principles of genetically encoded indicators. The
membrane voltage of a neuron provides information
about its synaptic inputs, cellular signal processing and
action potential output. Monitoring the ongoing opera-
tions of a neuronal circuit may therefore entail tracking
the time course of membrane potential fluctuations of
as many as possible of its constituent neurons. This may
be achieved with optical voltage indicators that trans-
form membrane potential fluctuations into changes in
light intensity 25,47,48. Alternatively, instead of directly
monitoring electrical activity, calcium indicators may
be used for the indirect detection of action potentials,
which are associated with a large increase in intracellu-
lar calcium concentration (arising from the opening of
voltage-gated calcium channels) in most cell types49,50.
Low molecular mass (LMM) organic compounds that
serve as calcium and voltage indicators have contributed
greatly to our understanding of circuits from both the
cellular and systems perspectives13,25,49.
The first prototypical protein-based fluorescent indi-
cators for calcium were published in 1997 (REFS 51,52),
and refinement and conceptual maturation over the fol-
lowing 15 years have resulted in tools that now enable
analysis of circuits at the level of genetically defined
cell classes. The design of genetically encoded calcium
indicators (GECIs) follows the model of LMM organic
calcium indicators, in which a fast calcium-binding mol-
ecule is structurally combined with a bright fluorescent
dye in a manner that results in a change in fluorescence
output upon binding of calcium53. Analogously, GECIs
involve the molecular fusion of a protein that under-
goes a conformational state transition in response to
calcium binding (such as calmodulin (CaM)51,52 or
troponin54) with a conformation-sensitive fluorescent
protein-based reporter component. The reporter com-
ponent can be either a single fluorescent protein55–57 or
a pair of fluorescent proteins that are suitable for Förster
resonance energy transfer (FRET)51,52,54 (FIG. 1A). GECIs
that exploit FRET yield opposing changes in donor
and acceptor fluorescence signals upon calcium bind-
ing, the ratio of which provides a measure of calcium
concentration that is independent of indicator concen-
tration, excitation light intensity and absorption in the
optical path. This ratio is therefore also insensitive to
fluctuations of these parameters.
As an alternative to FRET, the widely used GCaMP
family of GECIs55 makes use of circularly permuted fluo-
rescent proteins (cpFPs) fused with CaM and the M13
domain of a myosin light chain kinase (FIG. 1Ab)55,57,58.
cpFPs are susceptible to chromophore quenching as a
consequence of small conformational changes around
their interface with the attached calcium-binding pro-
tein. This, in turn, results in a large modulation of their
fluorescence output during increases in calcium concen-
tration55–57,59. Recent efforts in GECI enhancement have
focused on tuning for specific applications and optical
properties, such as calcium-binding constant59,60, subcel-
lular targeting 61, colour 62 and brightness59.
The first prototypical genetically encoded voltage
indicator (GEVI) was reported in 1997 (REF. 63), the same
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year as the first GECIs. However, optimization of GEVIs followed the model of previous non-genetic methods
to robustly work in mammalian neurons took another and in particular the use of LMM fluorescent dyes for
10 years64. Not surprisingly, the first GEVI conceptually tracking the conformational changes of ion channels

Box 1 | Microelectrodes or light?

Electrode arrays and optical methods are both mature technologies that allow one to record the activities of
thousands of individual neurons simultaneously in live mammals. Microelectrode arrays (MEAs) are used to record
action potentials from many individual cells as well as population signals (as local field potentials; LFPs). Population
signals can also be registered as electroencephalogram (EEG) or electrocorticogram (ECoG) with larger electrodes
mounted on the skull or directly onto the surface of the brain. Electrode-based and light-based methods also have
fundamental, but different, limitations. MEA recordings are typically obtained from spatially dispersed sets of neurons,
which limits the analysis of local circuit mechanisms. In addition, extracellular recordings provide perfect information
about action potentials, but are blind to subthreshold membrane potential fluctuations (including synaptic potentials)
and to silent neurons.
Optical methods, which use genetically encoded optical indicators (calcium-based or voltage-based), are non-invasive
and have excellent spatial resolution, but only to a tissue depth beyond which light scattering becomes prohibitive. With
some exceptions, optical recordings also have lower signal-to-noise ratios than electrical recordings. A comparison of the
advantages and limitations of these two approaches for single-cell resolution and population-level recordings is shown in
the table.

Feature MEAs (single cells) or LFP Genetically encoded optical

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.
Single-cell resolution level
Number of cells that can be
simultaneously recorded (best case)
Density (limit of spatial resolution)
Information content
Temporal resolution (best case)
Cell class identification
Spatial localization of signal (limit)
Recording depth
Feasibility to record from the same
cells over a week or longer
Population level
Spatial localization (limit)
Information content
Cell (class) identification or
addressability (a priori specification
of the cell class that is recorded
from)
electrodes, EEG (population
recordings)
~200
100 μm (due to tissue damage,
electrode wiring and volume
conduction of extracellular currents)
Action potentials and LFPs
Sub-millisecond range (practically
no limit)
Only possible indirectly and post hoc
Modest (exact absolute and relative
positions of recorded neurons
generally unknown)
Practically unlimited
Challenging, modest success
rate142–144
LFP: ~100 μm, frequency dependent
(volume conduction)
ECG and EEG: modest spatial
resolution and ambiguous in
localization of signals24
Extracellular currents from local
and distant sources; mainly synaptic
currents but also action currents24
Not possible in tissues in which
different cell types are intermingled
(although optoelectronic
device-based technologies may
eventually enable this)80,145
indicators (calcium or voltage)
~500 (in vivo)19–21
~1–5 μm (due to light scattering,
photon budget per time and spatial
bins)141
Calcium indicators: action potential
events (routinely achieved in vivo)
Voltage indicators: action potentials
and subthreshold potentials (not
routinely achieved in vivo)
Millisecond range (limited by indicator
properties)
Direct through specific genetic
targeting of indicator or marker protein
Microscopic level (due to light
scattering in tissue)
Depth in cortex routinely ~400 μm (up
to 700 μm)99,141; deeper with invasive
procedures80
Possible with high success rate21
Microscopic level (due to light
scattering in tissue, ~100 μm
in wide-field, non-confocal
epifluorescence imaging)
Calcium indicators: population action
potential activity
Voltage indicators: volume averaged
membrane potential (mainly synaptic
potentials)
Possible through specific genetic
targeting

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Genetically encoded calcium indicators (GECIs) Figure 1 | Genetically encoded optical indicators of
Aa neuronal activity.  A | Schematic depiction of calcium
YC2.60 indicators. Aa | Förster resonance energy transfer
CaM 2s
M13 (FRET)-based calcium indicators52,54 often use cyan
50 % fluorescent protein (CFP) and yellow fluorescent protein
Δ/R/R0
(YFP). Excitation of CFP with 440 nm light (blue arrows)
CFP YFP results in cyan (cyan arrows) and yellow (green arrows)
FRET fluorescence. Binding of Ca2+ (red circles) to a
calcium-binding domain (such as the calmodulin (CaM)–
FRET-based GECIs
1, 2, 5, 10 or 20 APs M13 complex shown here) increases the efficacy of FRET
between CFP and YFP51,52,54 and therefore increases the
Ca2+ fluorescence ratio (R) between these two fluorescent
Ab GCaMP3 proteins. Traces on the right show, for a representative of
this indicator class (YC2.60), the change of R (ΔR,
cpFP CaM
normalized to baseline, R0) when a pyramidal neuron
100% generates 1 (black), 2 (cyan), 5 (blue), 10 (green) or 20 (red)
ΔF/F0 action potentials (APs). The horizontal bars of
2s
corresponding colours indicate the time of AP firing.
Ab | Single fluorescent protein indicators of the GCaMP
M13 Ca2+ type55,59,62,85 incorporate a circularly permuted fluorescent
1, 2, 5, 10 or 20 APs
cpFP-based GECIs protein (cpFP). Binding of Ca2+ to CaM–M13 increases the
quantum yield of cpFP fluorescence (F). Traces on the right
show how a representative of this indicator class (GCaMP3)
Genetically encoded voltage indicators (GEVIs)
reports 1, 2, 5, 10 and 20 APs as a change of F (ΔF,
Ba normalized to baseline, F0). B | Voltage indicators.
FRET-based VSFPs
VSFP2.3 Ba | FRET-based voltage-sensitive fluorescent proteins
+ + – – (VSFPs) are engineered around a four transmembrane
segment (S1–S4) voltage-sensing domain64,65. The efficacy
S4

PM PM 1% ΔF/F0
S1
S2

S1
S2
S3
S4 3

of FRET increases upon plasma membrane (PM)

S

– – + + depolarization as the voltage sensor adopts its activated

200 ms
state. Traces on the right show optical recordings (changes
in baseline-normalized YFP fluorescence, ΔF/F0) from a
50 mV cultured hippocampal cell expressing a representative of
this indicator class (VSFP2.3). Three sweeps of optical
recordings (represented by the colours black, red and blue)
Bb Bc were acquired simultaneously with microelectrode
Single FP- or cpFP-based VSFPs Opsin-based voltage indicator recordings (shown superimposed with corresponding
colours). Data are adapted from REF. 48. Bb | Single FP/
+ + – – + + – –
cpFP-based VSFPs exhibit fluorescence quenching upon
S4

PM PM membrane depolarization69,70. Bc | Opsin-based sensors71

S1
S2

S1
S2
S3

N N
S4 3
S

– – + + – – + H+ + have been developed only recently. Their mechanism of

H+ action involves changes in fluorescence that are associated
with voltage-dependent protonation of a rhodopsin
molecule. However, the fluorescence of these GEVIs is dim
and they have not yet been tested in intact brain tissue.
Part A is adapted from REF. 77.

Nature Reviews | Neuroscience

(and originally the slow inactivation of the Shaker
potassium channel)63. Subsequent GEVI designs used
an isolated voltage-sensing domain fused with fluores-
cent proteins to track voltage changes more directly, and
are generally described by the acronym VSFP (voltage-
sensitive fluorescent protein)48,64,65. Presently, the most
successful GEVIs use a voltage-sensing domain derived
from the sea squirt Ciona intestinalis voltage sensor-con-
taining phosphatase (Ci‑VSP)48,64,66,67 (FIG. 1Ba,Bb).
VSFP2.3, a variant from the VSFP family, was the first
FRET-based GEVI that enabled the optical imaging of
action potentials and synaptic potentials in brain slices
and live mice48. An alternative molecular design (known
as VSFP3) uses a single fluorescent protein instead of
a fluorescent protein pair 68–70 (FIG. 1Bb). VSFP3s offer a
broad coverage of the colour spectrum and relatively
fast overall kinetics, but have signal amplitudes that are
smaller than those of the FRET-based VSFP2s. Current
efforts in the development of GEVIs are focused on opti-
mizing key specifications in existing sensor constructs,
such as voltage range, subcellular targeting properties,
response kinetics and colour 67–69. Design of sensors for
specific applications and exploring new mechanisms for
coupling membrane voltage to an optical readout are also
being undertaken. The most recent new design concept
for GEVIs is based on microbial opsins71 (FIG. 1Bc), but the
development of these molecules has not reached a stage
at which they can be validated in intact brain circuits.
Although there are clear conceptual reasons why
the use of GEVIs may be preferred over GECIs (such as
their capacity to report both synaptic input and action
potential output and their direct monitoring of the sig-
nal of interest), GECIs are more widely used because
they provide larger changes in measured light intensity

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© 2012 Macmillan Publishers Limited. All rights reserved


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.
NATURE REVIEWS | NEUROSCIENCE VOLUME 13 | O CTOBER 2012 | 691
and hence a higher signal-to‑noise ratio (SNR)13,72. In
addition to the specific classes of GECIs and GEVIs
mentioned and illustrated in this article, the complete
toolbox of genetically encoded indicators contains tools
for monitoring other important parameters such as the
concentration of synaptic transmitters and vesicular
release of transmitters73–76. These are powerful tools for
synaptic-level and subcellular-level studies, but most are
not widely used in circuit-level studies (with the excep-
tion of the synaptopHluorin73 class of indicators). The
decision to use a particular genetically encoded indi-
cator for a particular application may be facilitated by
the results of studies that have attempted a side‑by‑side
comparison of leading representatives of each indicator
class67,77,78.
Comparing GEVIs and GECIs with LMM indicator dyes.
In addition to the conceptual importance of cell class-
specific optical imaging, protein-based indicators allow
genetic ‘staining’ procedures that are both non-invasive
or minimally invasive and permanent. These features
make it straightforward to conduct imaging sessions
repetitively over days and weeks during the course of
normal physiological behaviour 8,8,21,79,80. This capacity for
chronic imaging opens the window for a large number of
studies on development, plasticity, functional regenera-
tion and slowly developing diseases that are associated
with abnormal wiring of neuronal circuits, which would
not be practical with stains that are typically obtained
with LMM indicators (which last only a few hours)81.
Much like corresponding organic dyes, GECIs add cal-
cium buffering capacity and GEVIs add electrical capac-
ity to the cells expressing these indicators. These effects
might adversely affect synaptic plasticity and integration,
action potential generation or long-term survival of
neurons expressing these indicators82,83. However, stud-
ies in which these issues were specifically addressed for
the GECI GCaMP3 (REF. 21,59) and the GEVI class of
VSFP2s48,82 did not reveal significant differences between
expressing and non-expressing brain tissue under exper-
imental conditions, whereby the indicators provided a
useful readout of neuronal activities. Furthermore,
for the GCaMP class of GECIs and the VSFP2 class of
GEVIs it has been demonstrated that mice expressing
these indicators survive and (in the case of transgenic
expression) propagate with no apparent deficits in their
functional integrity 48,84,85.
One of the main goals of GECI and GEVI develop-
ment over the past 15 years has been to increase their
SNR in biologically relevant experimental settings.
Nevertheless, organic calcium indicators provide an
excellent SNR for the detection of isolated action poten-
tials that is not readily matched by currently available
GECIs77. For instance, it has been noted that GCaMP3
detects smaller numbers of activated cells than LMM
organic dyes in experiments using physiological stimuli84.
This lower effective sensitivity (false-negative action
potential detection) could be due to various reasons.
The most important physical and chemical difference
between GECIs and LMM organic calcium indica-
tors is that GECIs have a slow forward rate of calcium
© 2012 Macmillan Publishers Limited. All rights reserved
REVIEWS
binding, which can bring them into an uncompetitive
position with regard to calcium access when compared
with endogenous fast calcium buffers83. Difficulties in
detecting isolated action potentials of GECIs may also
be explained by the lower indicator concentration that
is (currently) achieved with long-lasting (but slowly
developing) genetic expression as opposed to acute
dye-loading conditions. Although slow kinetics and low
capacity of the added calcium buffer reduces the SNR,
these factors also reduce potential interference with
calcium-dependent synaptic plasticity and integration.
Thus, there are arguments in favour of using GECIs
even under experimental conditions in which organic
dyes may provide a superior SNR. In addition, there are
also important experimental conditions (such as chronic
recordings) in which only GECIs are practical.
In experimental settings that require monitoring of
large numbers of cells, GECIs (like LMM organic cal-
cium indicators) provide a readout that is limited by
their relatively slow kinetics and that provides informa-
tion only on action potential events. Optical indicators
of membrane voltage that report synaptic and other sub-
threshold activities are preferable if this information is
desired25. Voltage signals are much faster than calcium
signals and their imaging therefore requires faster rates
of acquisition.
Furthermore, voltage indicators provide smaller
fluorescence changes than calcium indicators and the
number of indicator molecules that can be accommo-
dated in the cell membrane is low compared with that in
the cytosolic volume. These factors explain why voltage
imaging has a comparably low SNR13. However, VSFP2s
provide a SNR that is comparable to that of organic
voltage-sensitive dyes82 and, given their other inherent
advantages discussed above, they should perform better
in most voltage-imaging experiments at the population
level of cells. GEVIs cannot yet be broadly applied in
single-cell-level studies in live mammals; however, this
is also the case for LMM voltage-sensitive dyes.
Genetic targeting. As discussed above, the ability to tar-
get protein-based indicators to specific classes of cells
constitutes a key advantage of these molecules over
organic dye-based approaches. However, full realization
of this advantage will continue to require considerable
effort until ‘ready to go’ genetically modified animals are
widely available. Indicator genes can be introduced into
live animals by several non-invasive or minimally inva-
sive methods, including in utero electroporation, use of
viral vectors or generation of transgenic animals (FIG. 2).
In the simplest gene expression configuration, a
promoter or enhancer sequence naturally drives the
selective transcription of the indicator gene in a class
of cells (FIG. 2A–C), a system that has been widely used
for marking specific cell populations with fluorescent
proteins86,87. Transgenic mice that express GEVIs have
been generated by several laboratories85,88–90. However,
there are only a limited number of reports of neuron
class-specific expression of genetically encoded indica-
tors using this simple transgene configuration85,91. One
problem is that many cell classes are poorly defined
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a In utero electroporation Figure 2 | Delivery of indicator genes to specified

cell types in live animals.  a | In utero electroporation.
Enhancer/ Indicator A plasmid containing the indicator gene under the
promoter gene control of a strong enhancer or promoter sequence is
injected into the lateral ventricle of an embryo through
ATG pA Neocortical the wall of the uterus. The plasmid is electroporated into
pyramidal cells cells in the wall of the ventricle. Different cell classes
Pressure can be labelled by electroporation at different locations
injection during different developmental stages. Electroporation
Expression at mouse embryonic day 15.5, as illustrated on the right,
+ targets cells that migrate as pyramidal cells into layer
plasmid
– 2/3 of the cortex (the green colour indicates expression
50 V of voltage-sensitive fluorescent protein 2.3 (VSFP2.3),
Ventricle coronal section48). b | Viral vectors. The indicator gene is
assembled and packed into a virus particle, such as
b Viral vectors adeno-associated virus (AAV), in cultured cells
Vector plasmid transfected with vector and helper plasmids. After local
injection into the brain, the virus delivers the indicator
gene to nearby cells. Depending on the regulatory
sequences included, the indicator is expressed in
specific cell classes. The image on the right shows
AAV-helper expression of EGFP (enhanced green fluorescent
plasmid protein; the green patch, coronal section) in the cortex
Cultured cells Pressure following injection of a serotype 2/1 AAV with the
injection
AAV strong ubiquitous CAG promoter. c | Simple transgenic
animals. The indicator gene, driven by regulatory
sequences from a cell class-specific gene, is injected
c Simple transgenic animals into fertilized mouse ova, which are then transplanted
into a foster mouse. Offspring that have the gene
randomly inserted into their genome are propagated
Pronuclei
and display indicator expression in the selected cell
classes. The image on the right (parasagittal section)
shows the cerebellar cortex with GABAergic cells
Fertilized egg expressing EGFP (green)46 and granule cells expressing
EYFP (enhanced yellow fluorescent protein; red)87.
Foster mother d | Cre-loxP system. This requires two types of
Implant in genetically modified mouse lines: an ‘indicator’ mouse
uterus F1 line, in which the indicator gene is under control of a
F2 (usually) strong enhancer or promoter but inactivated by
a loxP flanked stop codon cassette (yellow triangle and
d Cre-loxP system: indicator and driver mice blue segement), and a ‘Cre driver’ mouse line, which
expresses the Cre-recombinase in specific cell classes.
Cre driver mouse Crossing these two lines yields offspring whereby
CRE Cre-recombinase removes the stop codon and activates
indicator expression in these selected cells. In the image
on the right84, the genetically encoded calcium indicator
× CRE GCaMP3 is expressed in cortical pyramidal cells.
e | Combining Cre-driver mice and viral vectors. The
STOP Indicator gene indictor gene is delivered by a viral vector (such as AAV)
in a Cre-recombinase dependent configuration. After
Indicator mouse carrying indicator gene in
local injection into the brain, the indicator is expressed
Cre-dependent configuration where virus infection and Cre-recombinase expression
intersect. The image on the right shows expression of a
red fluorescent protein in cortical interneurons of
e Combining Cre-driver mice and viral vectors parvalbumin-Cre driver mice151. With each strategy, the
protein can be expressed in a lifelong manner, but only
Indicator in genetically modified animals (c and d) is the gene
STOP gene passed to offspring. Viruses are also used in species,
Cre driver mouse
Pia including primates, for which transgenic model
development is more of a challenge. Scale bars
represent 1 mm (a,b), 100 μm (c,d) and 250 μm (e). The
AAV Pressure image in part a is reproduced, with permission, from
injection REF. 48 © (2010) Macmillan Publishers Ltd. All rights
reserved. The image in part c is reproduced from
REF. 152. The image in part d is reproduced, with
White matter permission, from REF. 84 © (2008) Society for
Neuroscience. Part e is reproduced from REF. 151.

Nature Reviews | Neuroscience

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© 2012 Macmillan Publishers Limited. All rights reserved


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.
NATURE REVIEWS | NEUROSCIENCE VOLUME 13 | O CTOBER 2012 | 693
with regard to unique regulatory sequences, and those
that have been identified often fail to yield sufficiently
high levels of protein expression92. These issues can be
addressed by using two or more promoters in such a
way that the indicator gene is only expressed where the
activation of these promoters intersects92.
The most widely used (and hence versatile) imple-
mentation of these combinatorial approaches is in
Cre recombinase-based strategies (FIG. 2D,E), in which a
(possibly) weak promoter drives the expression of Cre
recombinase, which in turn initiates the transcription
of an expression cassette that has a strong promoter 92,93.
For Cre recombinase-based strategies, the indicator gene
may be provided by a transgenic animal (called an indi-
cator or reporter mouse) or by a viral vector. Local injec-
tion of viruses provides a spatially restricted expression
pattern, which can be advantageous or limiting, depend-
ing on the experimental question being posed and the
spatial scale of the imaging approach. By contrast, genet-
ically modified mice that do not require local injection
of viruses can reproducibly express the indicator gene
in specific cell classes over the complete cortical space
(in fact, in the entire animal), thereby allowing access to
widely distributed neuronal computations.
In the future, it may be fruitful or even necessary to
define cell classes based not only on their gene expres-
sion profile but also on their functional role, such as their
activation during a defined behaviour 94,95 or their con-
nectivity 21,40,96. For these approaches, inducible expres-
sion systems in which transcription is reversibly turned
on (or off) in the presence (or absence) of an orally deliv-
erable substance (for example, tetracycline) can be used
to constrain the time window during which behaviour
triggers gene expression97,98.
Optical imaging technologies. The successful use of
these indicators relies on optimized optical instrumen-
tation. The most crucial feature for optical imaging of
circuit activities is the generation and sampling of large
numbers of photons from small volumes of tissue over
short time periods. This is because the optimal SNR is
directly proportional to the square root of the number
of detected photons. The generation of photons is lim-
ited by the photophysics of the indicator molecules,
each of which can provide photons at a limited rate
(1 per fluorescence lifetime) only until it is bleached
(determined by the quantum yield of bleaching)13,72.
Notably, in this context genetically encoded indicators
share a similar limitation at the single-cell level with
optogenetic actuators whereby molecular saturation sets
a upper limit to the amount of light-inducible photocur-
rent 13,94,99. If single-cell resolution is not required, for
instance because a functionally homogenous class of
cells is being examined, sampling over a larger number
of indicator molecules increases the yield of photons,
and therefore the SNR.
Imaging through brain tissue results in a loss of spa-
tial resolution due to light scattering, although this effect
can be reduced by using laser-scanning microscopy
(LSM), in which the origin of light is known from the
position of the illumination. In the case of 2P excitation,
© 2012 Macmillan Publishers Limited. All rights reserved
REVIEWS
this spot (also known as the point spread function) is less
than 1 μm in diameter even at depths of a few hundred
micrometres, and cellular resolution imaging is possible
at tissue depths of up to about 800 μm100,101. Although the
high spatial resolution achieved by 2P imaging 100 cre-
ates the possibility of routinely performing in vivo opti-
cal imaging with cellular resolution, it should be noted
that in LSM photons are sampled from only one position
in the image at a time. In wide-field imaging using a
charge-coupled device or complementary metal-oxide
semiconductor sensors, photons are sampled simul-
taneously from all object points (pixels in the image),
therefore yielding a much better photon budget and
SNR. Unfortunately, this increased SNR comes with
blurred spatial information due to light scattering and
out‑of‑focus light. In fluorescence imaging, the latter can
be reduced by limiting the expression of indicators to
cells of interest 101.
Considering these strengths and limitations of LSM
and wide-field illumination, several research groups
have pursued alternative imaging strategies with the
goal of finding a better compromise between spatial
resolution and SNR. The most promising of these
include wide-field 2P excitation (using, for example,
temporal focusing) 102–104, sheet illumination 105 and
light patterning techniques103. These new instrumen-
tal solutions have already proven advantageous when
using optogenic control tools and classical fluorescent
indicators103,104, and will play an increasing role in many
circuit-centric imaging approaches.
Applications
To what extent have genetically encoded indicators of
neuronal activity and the associated genetic and imag-
ing technologies already been put into practice for the
analysis of neuronal circuits? In the following sections,
I provide an overview of some recent pioneering exam-
ples which show that application of these concepts and
methods are already yielding fruit.
Analysis of defined cell classes at the population level.
The first transgenic mouse line expressing a GECI
(GCaMP2) in specific types of neurons was reported in
2007 (REF. 106) and was used to characterize the function
of synapses formed by parallel fibres in the molecular
layer of the cerebellum107,108 (FIG. 3A). As indicator expres-
sion in the cerebellar cortex was limited to cerebellar
granule cells (FIG. 3Aa), calcium transients measured
within volumes on the scale of tens of micrometres in
brain slices (FIG. 3Ab,Ac,Ae) and in vivo (FIG. 3Ad) could be
attributed to the 0.2 μm-thick granule cell axons (paral-
lel fibres) and their presynaptic specializations (formed
every 5–10 μm along their path).
In a more recent study, GCaMP3 was expressed in
a genetically specified population of zebrafish spinal
motor neurons to analyse the development of the spinal
central pattern generator109 (FIG. 3B). Analysis of the spon-
taneous activities of single motor neurons established
the developmental emergence of synchronization within
the neurons on one side of the spinal cord and across the
two sides. FIGURE 3Bb,Bc illustrates the phase relationship
REVIEWS

between the left and right motor neuron pools, exem- The first reliable population voltage signals from genet-
plifying a situation in which comparative population ically targeted neurons in live mice were imaged using
analysis of selected cell classes is sufficient to reveal a second-generation FRET-based VSFPs48. VSFP-based
physiologically relevant activity pattern. This study is imaging in cortical brain slices resolved synaptic poten-
also particularly remarkable as it combined the use of tials and (with a lower SNR) action potentials from single
an indicator protein with the use of an actuator protein cells without the need to average over repeated trials48.
to characterize neuronal circuit physiology. FIGURE 3C illustrates VSFP-based voltage imaging in a hip-
pocampal brain slice under experimental conditions in
which the activity of pyramidal cells was synchronized by
Aa Ab Stimulation 0.4% Stimulation Ad bath application of a glutamate receptor agonist.
ml electrode electrode
gl Taken together, these examples show how geneti-
ml cally encoded indicators of activity can be particularly
useful for examining the workings of circuits in which
0.0% Caudal 0.3 mm genetically well-defined populations of neurons act in
P Ac synchrony, such that population signals reveal an activ-
0% 15% Ae
1%
ity pattern that represents a physiologically relevant
feature common to individual neurons. As GECIs and
gl 500 ms
GEVIs that produce different colours of fluorescence
100 μm 1 mm
are available62,69,78, these approaches may be extended to
study local interactions between multiple classes of cells.
Ba Ca Cb This approach will facilitate the examination of local
CA1 CA1
circuit mechanisms underlying gain modulation110–112,
dynamic balance of excitation and inhibition113, and
CA3 rhythmic activity 6.
CA3 200 μm 200 μm

Bb Cc Organization of representations in anatomical space.

LFP 50 μV
ΔF/F
50%
ΔF/F
10 s Donor 0.5%
Bc Acceptor
ΔF/F ΔR/R
1%
50% Ratio
60 s 2s 200 ms
Figure 3 | Population responses from genetically targeted cell classes. 
Nature Reviews Neuroscience
A | Cerebellar granule cells. Aa | This shows immunohistochemical staining|against the
genetically encoded calcium indicator GCaMP2. GCaMP2 expression (brown) in the
granule cells of the cerebellar cortex of mice is under the control of the regulatory
sequences of the gene KCNC1. The image shows the molecular layer (ml), Purkinje cell
layer (P) and the granule cell layer (gl); arrowheads point at granule cell bodies and arrow
points at their axons (the parallel fibres). Ab,c | This shows a transmission image of a
transverse cerebellar slice (Ab) and change in GCaMP2 (Ac) fluorescence (ΔF/F,
indicating a transient elevation of parallel fibre calcium concentration) induced by
stimulation of the molecular layer (10 stimuli delivered at 100 Hz). Asterisk indicates tip
of stimulation electrode. Ad | This shows the corresponding calcium signal in parallel
fibres imaged in vivo from the surface of the cerebellar cortex. Ae | This shows the
GCaMP2 parallel fibre calcium signal in response to eight stimuli (arrows) delivered at
10 Hz. Data from REFS 85, 106. B | Motor neurons in the zebrafish spinal cord. Ba |
Fluorescence image of the spinal cord of a zebrafish embryo in which a population of
motor neurons express GCaMP3 (dorsal view). Bb,c | Motor neuron calcium signals taken
from the left and right sides of the spinal cord (blue and red traces, respectively) at fast
(Bb) and slow (Bc) timescales. The patterns of calcium elevations indicate the activity and
phase difference between left and right motor neuron pools. Data kindly provided by the
authors of REF. 109. C | Hippocampal pyramidal cells. Confocal (Ca) and wide-field (Cb)
image of a hippocampal slice prepared from a mouse that was electroporated in utero
with VSFP-Butterfly 1.2 (REF. 154 ). Cc | Optical and local field potential (LFP) recordings
in a slice that was treated with 60 nM kainate to induce spontaneous population bursts
(optical recording area marked by red circle in Cb). Traces show LFP recordings
(electrode indicated in Cb), optical signals of the donor fluorescent protein and the
acceptor fluorescent protein, and the acceptor:donor ratio at slow (2 s) and fast (200 ms)
time resolutions. The increase in the acceptor:donor ratio represents an increase in the
membrane voltage. Data were acquired using methods described in REFS 48,154. Parts
Aa–d are modified, with permission, from REF. 85 © (2005) Oxford University Press.
A general strength of optical imaging is that it can map
recruitment patterns within anatomical space. Cell class-
specific targeting of indicators can help to dissect overlay-
ing or intermingled maps. Among the first realizations
of this strategy were studies that required the imaging of
macroscopic sensory maps of the mouse olfactory bulb
(FIG. 4A). SynaptopHluorin, a genetically encoded indicator
of synaptic release, was used to generate odour maps that
represent the presynaptic activity of olfactory nerve termi-
nals91 (FIG. 4Ab), and selective genetic targeting of GCaMP2
to neurons that are synaptically excited by olfactory nerves
has enabled the imaging of postsynaptic odour maps of
the olfactory bulb114,115 (FIG. 4Ac). The calcium imaging
approach showed that the response dynamics for a given
odour differed between olfactory bulb glomeruli (FIG. 4Ad).
The use of macroscopic imaging approaches to under-
stand odour representation in the olfactory bulb glomeru-
lar layer has been justified by the results of high-resolution
imaging studies that have revealed no heterogeneity in
responses within glomeruli, at least under the conditions
studied to date116. Macroscopic maps of sensory represen-
tation have also been shown in a proof-of-principle study
with GEVIs in the mouse barrel cortex, in which expres-
sion of VSFP2s was restricted to layer 2/3 pyramidal cells
and whisker deflection-induced responses of this selected
cell class were mapped48.
The visual cortex is a classic example of a brain struc-
ture in which optical imaging was used to establish rep-
resentations at multiple anatomical scales resulting in
fundamental discoveries, such as the pinwheel-like map
of preferred stimulus orientations in the primary visual
cortex of higher mammals117–121. The obvious follow‑up
to these classic findings is to examine whether specific
cell classes have specific maps. To resolve this question,
initial experiments combined the use of transgenic mice

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© 2012 Macmillan Publishers Limited. All rights reserved

 REVIEWS

in which inhibitory neurons were tagged with green
fluorescent protein with non-genetically encoded cal-
cium indicators122,123 and post-hoc immunohistochem-
istry 45,124. More recent studies used targeted expression
of GCaMP3 in cortical pyramidal cells84 and extended
the primary visual map to higher visual cortical areas125.
A n o t h e r r e c e nt l a n d m a r k s t u d y 8 0 u s e d
GCaMP3‑based imaging in combination with spatial
navigation in a virtual reality setting to map the spatial
distribution of hippocampal place cells (FIG. 4B). This study
revealed little or no correlation between the anatomical
distance between place cells and the distance between
the place fields that they represent 80 (FIG. 4Bc). Spatially
intermingled representations were also found in a recent
study whereby GCaMP3 was used to investigate the
formation of representations in the motor cortex while
mice learned to detect objects with their whiskers and
report detection by motor behaviour 21. For this study, as
for the other studies in awake behaving mice, repeated
imaging sessions over several days or weeks were nec-
essary, therefore highlighting this unique advantage of
genetically encoded indicators. Such a detailed analysis
of cellular-scale representations will require 2P imaging
at single-cell resolution, at least until methods to identify
individual cells using combinations of coloured geneti-
cally encoded indicators markers (in a manner similar

Macroscopic level

CCD 8%
Aa Ab Ac Stimulus Ad 1 3
6
SpH or 4 ΔF/F
GCaMP2
Ca2+ signals
5 2
0%
Fluorescence
excitation

Odour SpH GCaMP2 1 s ΔF/F

stimulus 4%

Microscopic level

Ba Bb 2 Bc
Scanner 1
5
Ti:S 8 10
7 6 9
VR display
PMT

4 3
ΔF/F 20 μm
50% 9

6 180 cm

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.
3
20 μm
2 0 cm
Floating ball treadmill Position on linear track Localization of place cells and colour-
2s coded position of their place fields
Figure 4 | Mapping representations onto anatomical space.  A | Mapping odour maps in the olfactory bulb at the
macroscopic level. Aa | Wide-field macroscopic charge-coupled device (CCD) imaging of the surface of the olfactory
Nature Reviews bulb
| Neuroscience
in mice expressing the presynaptic activity indicator synaptopHluorin (SpH) in olfactory nerve terminals and in mice
expressing the genetically encoded calcium indicator GCaMP2 in cells synaptically driven via the olfactory nerve.
Ab | Presynaptic odour (hexanone) map revealed by SpH signals. Ac | Postsynaptic maps revealed by GCaMP2. Ad | The
upper panel shows the glomerular odour response map evoked by propanal. The lower panel shows the response time
courses of the six different glomeruli indicated in the response map (traces for glomerulus 1 to 6 from top to bottom). The
green horizontal bar indicates the time of odorant delivery. B | Mapping neuronal representations at the microscopic level.
Ba | Experimental setup for cellular-resolution imaging of mice navigating in a virtual environment. Two-photon images are
obtained by sampling fluorescent light with a photomultiplier (PMT) while a focused infrared laser beam (Ti:S) scans neurons
in the brain of an awake mouse undertaking a navigation task. The mice walk on a floating ball treadmill that is linked to a
virtual reality (VR) display such that the virtual environment (blue vertical bars) moves according to the mouse’s intention to
navigate within that virtual space. Bb | Two-photon images of hippocampal CA1 cells. Cells activated at specific places in a
virtual environment are highlighted in red and numbered. GCaMP3 signals (ΔF/F, change in baseline normalized
fluorescence) for four of the numbered cells with activation (highlighted by the red portion of the trace) occurring at
specific positions along the virtual linear track. Bc | Place cells colour-coded according to the location of their place fields.
Note that the place field position does not correlate with anatomical position. Scale bars in Ab–Ad represent 500 μm. Data
in part B are adapted from REF. 80 with kind permission of the authors. Part Ab is modified, with permission from REF. 91 ©
(2004) Elsevier. Parts Ac,Ad are modified, with permission, from REF. 114 © (2009) the American Physiological Society.

NATURE REVIEWS | NEUROSCIENCE VOLUME 13 | O CTOBER 2012 | 695

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REVIEWS

to the cell identification strategy achieved using the
Brainbow approach126) are established.
Neuronal circuit dynamics. The term neuronal circuit
dynamics refers to the evolution of activity patterns in
space and time. Monitoring these patterns captures the
generation and propagation of what has been referred
to as the neuronal code. It is likely that there are differ-
ent coding schemes for different brain functions: some
might be based on the average activity of fixed ensembles
of neurons, whereas others might involve dynamic for-
mation of cell assemblies14. Higher cognitive functions
involve interactions among circuits that are widely dis-
tributed in the brain, with each local circuit displaying
its own cellular dynamics4,6,23,127.
Voltage imaging has for many years been considered
a promising approach to assess these multiple scales of
structural and functional organization at high temporal
resolution25. Proof-of-principle experiments for macro-
scopic level voltage imaging using GEVIs are illustrated

Macroscopic circuit dynamics

Aa CCD 1 Ab Ac
Rostral 2
Acceptor Medial 1
CCD 2

–10 ms 10 ms 30 ms 50 ms
Donor
Ad
Excitation 1

2 70 ms 90 ms 110 ms 130 ms
0.4%
100 ms 0% ΔR/R 0.6%

Microscopic circuit dynamics

Ba Reward Reward Bb Bc
2 Trial preference ( right, left)
1 1

selectivity index
Correct Correct

Trial-type
left right 3
choice choice 0

Delay
50 μm

period
–1
50 μm

Cue
Bd
offset Correct right trials Correct left trials
1
Cue
period
2
50 cm

ΔF/F
Trial 10 s 100%
start 3
Maze 1 Maze 2
Figure 5 | Neuronal circuit dynamics.  A | Macroscopic level analysis of circuit dynamics. Aa | Experimental configuration
Nature Reviews | Neuroscience
for voltage imaging using a Förster resonance energy transfer (FRET)-based voltage-sensitive fluorescent protein
(VSFP-Butterfly 1.2) in mice. The fluorescence emission from the VSFP is split by a dichroic mirror into donor and acceptor
colour channels and imaged by two synchronized charge-coupled device (CCD) cameras. Single whiskers are mechanically
stimulated by a piezoelectric device. Ab | Transcranial view through the thinned bone of the somatosensory cortex
contralateral to the stimulated whisker from a mouse electroporated in utero with VSFP-Butterfly 1.2 (REFS 48,153,154).
Anatomical orientation as indicated (scale bar represents 2 mm). Ac | A series of images representing the population voltage
response to a brief deflection of a whisker at time zero. Note the spread of activation from region 1 to region 2 as indicated
by the arrow. The area imaged is marked by a rectangle in panel Aa. Scale: 1 mm; average over 32 trails. Ad | Optical VSFP
signals sampled from two regions of interest indicated by white circles in Ac. Six traces from repeated measurements are
superimposed to demonstrate single trail resolution and robust response pattern. B | Microscopic level analysis of circuit
dynamics. Ba | Schematic of the two versions of a T‑maze presented to a mouse using a virtual reality display8. The mouse
learns to associate different cues with left and right reward locations. Bb | Image of neurons in posterior parietal cortex
layer 2/3 expressing the genetically encoded calcium indicator GCaMP3. Bc | Cells activated during each choice are
indicated by different colours, with red and blue indicating right and left choice preferences, respectively. Bd | GCaMP3
fluorescence traces portraying activity (Ca2+ transients, green) for the three example cells encircled in Bb. Cells 1 and 2 are
preferentially activated during choices to turn right early and late in the trial, respectively. Cell 3 is activated during the
choice to turn left. Part B modified, with permission, from REF. 8 © (2012) Macmillan Publishers Ltd. All rights reserved.

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© 2012 Macmillan Publishers Limited. All rights reserved

 REVIEWS

in FIG. 5A. In these experiments, a pair of charge-cou-
pled device cameras imaged the ratiometric optical
signal of a VSFP as a single whisker of a mouse was
deflected. The optical signal readily resolved the spread
of stimulus-induced depolarization from an initiation
site to other cortical areas (FIG. 5Ac), as well as spon-
taneous ‘ongoing’ activity 128,129,154. Similar macroscopic
level circuit dynamics have previously been obtained
with organic voltage-sensitive dye imaging 26,32,130–133, but
the VSFP reports changes in local membrane voltage
within a selected cell population (layer 2/3 pyramidal
cells, in the above-illustrated case).
At the microscopic level, circuit dynamics are typi-
cally observed as rhythmically synchronized or sequen-
tial activation of individual cells8–10,80,134–136. A recent
study that used GCaMP3 and single-cell resolution
imaging (FIG. 5B) demonstrated that neurons in the pos-
terior parietal cortex (PPC) were sequentially activated
as a mouse decided which of two paths (left or right)
in a virtual environment to take while being cued as
to which path will lead to a water reward8. PPC cells
that were active in association with either “go left, as
cued” or “go right, as cued” decisions were intermingled
at the microcircuit length scale (<100 μm) (FIG. 5Bb–Bd).
The two behavioural alternatives were associated with
distinct sequences of cell activity. This study advanced
beyond the relatively static maps of representations stud-
ied in previous efforts at single-cell level in vivo calcium
imaging, thereby entering the field of circuit dynamics.
In the future, similar studies might investigate the expe-
rience-driven dynamic formation of cellular assemblies
and the recall of previously formed assemblies. Such
studies will require behaviourally triggered tagging of
specific cell populations with indicator proteins95,137.
Stability and formation of representations. The term
neuronal circuit dynamics used above referred to activ-
ity patterns on the timescale of neuronal computation
(milliseconds to seconds). However, circuit features such
as representations of specific sensory and behavioural
parameters might change over a much longer timescale.
These issues can only be examined by experiments that
involve repetitive imaging sessions over days and weeks
(chronic experiments).

a
Day 1 Day 12 Day 19
6 4 4

4
ΔR/R (%)

2 2
2

0 0 0

-2
0 20 40 60 80 0 20 40 60 80 0 20 40 60 80
Time (s) Time (s) Time (s)

b Cell 1 Cell 2 Cell 3

8 14
Day 1 Day 1 5 Day 1
7 12
Day 3 Day 3 Day 12
6 4
10 Day 19
5 Day 5 Day 5
ΔR/R (%)

8 3
4
6
3 2
2 4
1
1 2
0 0 0

Figure 6 | Chronic imaging in vivo using GECIs.  An example of repetitive imaging sessions over several days that are
made possible by the use of genetically-encoded indicators. In this study, the calcium indicator TN‑XXL was expressed in
Nature Reviews | Neuroscience
layer 2/3 pyramidal cells of mouse primary visual cortex (V1) using in utero electroporation. Moving gratings with
different orientations (as indicated at the top of the figure) were displayed on a screen (during the time periods marked
by the vertical grey stripes) that was in front of the mouse on the side contralateral to the imaged V1. The gratings were
presented during the time periods marked by the vertical grey stripes. a | Visually evoked calcium transients (indicated
by a change in baseline normalized fluorescence ratio (ΔR/R) between yellow and cyan fluorescent proteins) in the same
neuron (shown in inset) on days 1, 12 and 19. b | The three graphs show orientation tuning curves (number of evoked
action potentials as indicated by the amplitude of the calcium transient versus the orientation of the stimulus) from three
individual neurons obtained on different days after the start of the experiment. Note the stable tuning over time in all
three cells. This type of experiment is greatly facilitated by the long-lasting labelling feature of GECIs, whereas low
molecular mass indicators leak out of cells a few hours after loading, thereby requiring impractical re‑staining
procedures81. Modified, with permission from REF. 79 © (2010) Macmillan Publishers Ltd. All rights reserved.

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REVIEWS

The unique advantage of GECIs for chronic experi-
ments was specifically demonstrated for the first time
in a study using TN-XXL79,81. This experiment (FIG. 6)
addressed the questions of whether the orientation
selectivity of individual layer 2/3 pyramidal cells in the
mouse primary visual cortex (V1) is stable over periods
of weeks79. The observed stability of tuning curves is
consistent with a hard-wired single neuron-level repre-
sentation of preferred stimulus orientation in V1.
A very recent study (using chronic 2P GCaMP3
imaging), by contrast, found that representation of
learned sensory and behavioural parameters by layer 2/3
pyramidal cells in mouse motor cortex are both plastic
and variable between repeated sessions21. This experi-
ment required researchers to follow the activity of large
numbers of individual neurons while a mouse was learn-
ing a sensory–motor detection and perception-reporting
task in several sessions over 1–2 weeks, a procedure that
could not have been achieved with previous method-
ologies based on LMM optical indicators. Interestingly,
this study revealed that population representations are
considerably more stable than single-neuron represen-
tations. Thus, this most recent study is (at the time of
writing this review) probably also the most impressive
example of the unique experimental and conceptual
advances offered by genetically encoded activity indica-
tors for the analysis of neuronal circuits.
Conclusions and future perspectives
Genetically encoded probes of neuronal activity have been
received with a mixture of curiosity and scepticism — but
also with high expectations — by neuroscientists inter-
ested in neuronal circuits. Some careful reservation was
indeed reasonable, as these probes have only very recently
begun to achieve a level of performance that is compara-
ble to established LMM organic indicators. Acceptance
of this new methodology has been further delayed by
the required crossing of borders between traditional
disciplines to merge genetic, electrophysiology and imag-
ing techniques. Now that these initial hurdles are being
overcome, the next big challenge is to put the potential of
these tools into general practice for the targeted analysis
of specific cell classes. The anticipated broad application
of genetically encoded indicators will stimulate further
improvement of already existing indicator classes and
motivate the development of new indicator classes based
on innovative methodologies138 and novel mechanisms.
The wish list for the next generation of GECI and GEVIs
includes red and infrared variants with increased photo-
stability, faster response kinetics and increased effective
sensitivity. In the future, I anticipate that the advantages of
genetically encoded indicators will surpass the traditional
use of LMM organic dyes for many applications and, in
particular, for circuit-centric analysis of brain functions.
This Review focused on monitoring neuronal activ-
ity; however, these observational approaches can only
reveal correlations with behaviour, and experimental
intervention is required to substantiate causal relation-
ships such as sufficiency and necessity 14,28,71,114. This need
motivates efforts to combine genetically encoded indica-
tor proteins with their methodological and conceptual
siblings: the light-activated ion channels and electrogenic
pumps used as optogenetic control tools39,139,140. Other
tasks that have yet to be accomplished include integra-
tion of the approaches described here with theoretical
approaches43,141, simulation of large-scale networks and
accumulation of structural circuit data142–144. Indeed, the
combination of these complementary efforts is beginning
to take place at major neuroscientific research centres142.
Palettes of genetically encoded indicators, once available
in a ready‑to‑go form in genetically modified animals84,
will ultimately become as familiar in the laboratory of
the neurophysiologist as the electrodes and electronic
instruments at their microscope. The prospects look
good for light-based neurophysiology 72 to initiate an age
of enlightenment in neuroscience.

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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.
Competing interests statement
The author declares no competing financial interests.
FURTHER INFORMATION
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