Chapter 5 - Neural activity imaging with genetically encoded calcium indicators
Introduction
One of the primary challenges of neuroscience is to link complex neural phenomena to the structure and function of their composite neural circuits. Addressing this problem requires a thorough understanding of patterns of neural activity and the ability to relate this to physiological processes, behavior, and disease states. An essential step toward this goal is the simultaneous recording of neural activity in large, defined populations, ideally in intact circuitry.
Traditional electrophysiological approaches provide excellent sensitivity and temporal resolution (Scanziani and Hausser, 2009) but are limited in the number of cells that can be recorded simultaneously. More importantly, assigning this activity to specific cells is quite difficult (and impossible for more than a few cells at a time), limiting the ability to create high-resolution circuit maps. Modern fluorescence imaging techniques (Svoboda and Yasuda, 2006), combined with high-quality fluorescent indicators (both small molecules and proteins), can potentially overcome these limitations. There is a rapidly growing toolkit of reagents that transduce changes in neural state (e.g., membrane potential or calcium ion flux, [Ca2 +], following action potentials (APs) or synaptic input), to fluorescence (or in some instances, luminescence, etc.) observables (for reviews, see Miyawaki, 2011, Palmer et al., 2011). Protein sensors are genetically encoded and can thus be used to label large populations of defined cell types and/or subcellular compartments (Borghuis et al., 2011, Dreosti et al., 2009, Mittmann et al., 2011, Shigetomi, Kracun and Khakh, 2010, Shigetomi, Kracun, Sofroniew and Khakh, 2010), unlike small molecule dyes, whose delivery and targeting can be problematic (Hendel et al., 2008, Shigetomi, Kracun and Khakh, 2010, Shigetomi, Kracun, Sofroniew and Khakh, 2010). In principle, genetically encoded sensors allow long-term measurements of activity in vivo, simultaneously across a neural population. Since the creation of the first generation of genetically encoded calcium indicators (GECIs), a decade ago (Miyawaki et al., 1997, Romoser et al., 1997), their performance has been iteratively optimized for applications in neurophysiology (as well as in other excitable cells such as cardiomyocytes (Tallini et al., 2006); recently, GECIs have also been used to monitor Ca2 + transients in nonexcitable cell types, such as astrocytes (Gourine et al., 2010, Shigetomi, Kracun and Khakh, 2010). The culmination of these efforts has led to activity measurements of defined neuronal populations in awake, behaving animals (Chiappe et al., 2010, Dombeck et al., 2010, Lütcke et al., 2010, Muto et al., 2011, O'Connor et al., 2010, Seelig et al., 2010, Tian et al., 2009).
In neurons, APs generate small calcium transients that can occur over a wide range of frequencies. To enable quantitative measurements of neural activity, one, in principle, desires a sensor with fast rise and decay kinetics, broad dynamic range, and calcium affinity appropriate for the cells in question (Hires et al., 2008). In addition, the basal brightness of the sensor should be high enough to permit identification of positive cells and improve the signal-to-noise ratio (SNR) of baseline measurements. Improved brightness also facilitates imaging with lower excitation power, important for minimizing indicator photobleaching and illumination phototoxicity.
In this chapter, we highlight recent progress in GECI design, optimization, and testing protocol standardization. We provide guidelines for selecting the appropriate GECI for a given biological application and discuss the remaining hurdles to perfect chronic, robust neural activity imaging in vivo.
Section snippets
GECI development and neuroscience applications
The general paradigm of GECI design is to fuse a calcium-binding domain (e.g., Calmodulin, CaM, or Troponin C, TnC) to one or two fluorescent proteins (FPs). In single-FP GECIs, the fluorescence intensity of a circularly permuted or split FP is modulated by calcium binding-dependent changes in the chromophore environment (Baird et al., 1999, Nagai et al., 2001, Nakai et al., 2001). In two-FP GECIs, calcium binding allosterically modulates the relative donor–acceptor emission spectra through
Case study: Rational design and systematic screening of GCaMP3
To develop GECIs with properties optimized for a particular application, the appropriate combination of intrinsic GECI parameters should be matched to the extrinsic factors of the system studied. Intrinsic GECI parameters include sensor affinity, kinetics, dynamic range, brightness, expression level, fluorescence properties, and independence from endogenous interference (for recent reviews, see Hires et al., 2008, Tian et al., 2011). Extrinsic parameters include the size, speed, time-course,
Choosing the best GECI for a given application
For end users, choosing the most appropriate GECI at the start of a project is paramount, since experimental optimization requires significant investments in time and resources. GECI selection will also influence many other choices, for example, light source, filters, camera, image analysis algorithms, etc. A good rule of thumb is to characterize several GECIs in the context of a specific application, as they each have different strengths and weaknesses. Here, we discuss several practical
Imaging neural activity with GECIs in vivo
Through iterative cycles of optimization, GECIs have been improved to the point that they are useful for in vivo neuronal imaging. State-of-the-art GECIs, such as D3cpV, YC3.60, YC-Nano, GCaMP-HS, and GCaMP3, have been widely used to report neural activity in living zebrafish (Muto et al., 2011), worms (Tian et al., 2009), flies (Cheng et al., 2010, Chiappe et al., 2010, Heim et al., 2007, Seelig et al., 2010, Tian et al., 2009), and mice (Dombeck et al., 2010, Lütcke et al., 2010, Mittmann et
Outlook
The primary focus of current GECI engineering efforts is to further increase the SNR for robust detection of low firing rates (ideally single APs) in a variety of systems in vivo. Engineering high-affinity sensors for probing small stimuli while preserving fast kinetics is challenging (Mank et al., 2006). Further optimization of the GCaMP, Cameleon, and TnC sensor formats will undoubtedly improve signals in the short term. Alternatively, novel GECI scaffolds, with different (perhaps faster)
Reagent availability
GCaMP3 plasmid DNA (CMV promoter for expression in mammalian cells) is available from Addgene (addgene.org): #22692. A membrane-fused version (Lck) is also available: #26974. Live AAV virus and pAAV constructs for both GCaMP3 and a Cre-dependent FLEX-GCaMP3 are available from the U Penn Vector Core, driven by a synapsin-1 promoter (http://www.med.upenn.edu/gtp/vectorcore/Catalogue.shtml). Drosophila strains expressing GCaMP3 under UAS control are available from the Bloomington Stock Collection
Note added in proof
Recently, blue- and red- shifted single-FP GECls have been demonstrated (Zhao et al., 2011).
References (74)
- et al.
Crystal structures of the GCaMP calcium sensor reveal the mechanism of fluorescence signal change and aid rational design
The Journal of Biological Chemistry
(2009) - et al.
The role of the TRP channel NompC in Drosophila larval and adult locomotion
Neuron
(2010) - et al.
Walking modulates speed sensitivity in Drosophila motion vision
Current Biology
(2010) - et al.
Automated correction of fast motion artifacts for two-photon imaging of awake animals
Journal of Neuroscience Methods
(2009) - et al.
Genetically encoded indicators of cellular calcium dynamics based on troponin C and green fluorescent protein
The Journal of Biological Chemistry
(2004) - et al.
Ca2 + buffering and action potential-evoked Ca2 + signaling in dendrites of pyramidal neurons
Biophysical Journal
(1996) - et al.
Autofluorescent proteins with excitation in the optical window for intravital imaging in mammals
Chemistry & Biology
(2009) - et al.
Genetic dissection of neural circuits
Neuron
(2008) - et al.
A FRET-based calcium biosensor with fast signal kinetics and high fluorescence change
Biophysical Journal
(2006) - et al.
Automated analysis of cellular signals from large-scale calcium imaging data
Neuron
(2009)
Neural activity in barrel cortex underlying vibrissa-based object localization in mice
Neuron
Ca2 + indicators based on computationally redesigned calmodulin-peptide pairs
Chemical Biology
Design and application of genetically encoded biosensors
Trends in Biotechnology
Fluorescent protein FRET: The good, the bad and the ugly
Trends in Biochemical Sciences
Detection in living cells of Ca2+−dependent changes in the fluorescence emission of an indicator composed of two green fluorescent protein variants linked by a calmodulin-binding sequence. A new class of fluorescent indicators
Journal of Chemical Biology
Tropomyosin localization reveals distinct populations of microfilaments in neurites and growth cones
Molecular and Cellular Neurosciences
Mapping and manipulating neural circuits in the fly brain
Advances in Genetics
Principles of two-photon excitation microscopy and its applications to neuroscience
Neuron
Spike inference from calcium imaging using sequential Monte Carlo methods
Biophysical Journal
Structural basis for calcium sensing by GCaMP2
Structure
Alternative life histories shape brain gene expression profiles in males of the same population
Proceedings of the Royal Society B
Circular permutation and receptor insertion within green fluorescent proteins
Proceedings of the National Academy of Sciences of the United States of America
Adeno-associated viral vectors for mapping, monitoring, and manipulating neural circuits
Human Gene Therapy
Imaging light responses of targeted neuron populations in the rodent retina
The Journal of Neuroscience
Dissecting a circuit for olfactory behaviour in Caenorhabditis elegans
Nature
Filtering of visual information in the tectum by an identified neural circuit
Science
Activation of cerebellar parallel fibers monitored in transgenic mice expressing a fluorescent Ca2 + indicator protein
The European Journal of Neuroscience
Functional imaging of hippocampal place cells at cellular resolution during virtual navigation
Nature Neuroscience
A genetically encoded reporter of synaptic activity in vivo
Nature Methods
Troponin C in brain
Nature
Astrocytes control breathing through pH-dependent release of ATP
Science
Functional fluorescent Ca2 + indicator proteins in transgenic mice under TET control
PLoS Biology
Encoding gender and individual information in the mouse vomeronasal organ
Science
Improved calcium imaging in transgenic mice expressing a troponin C-based biosensor
Nature Methods
Fluorescence changes of genetic calcium indicators and OGB-1 correlated with neural activity and calcium in vivo and in vitro
The Journal of Neuroscience
Reporting neural activity with genetically encoded calcium indicators
Brain Cell Biology
Spontaneous network activity visualized by ultrasensitive Ca(2 +) indicators, yellow Cameleon-Nano
Nature Methods
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Current address: Department of Biochemistry and Molecular Medicine, School of Medicine, University of California, Davis, 95817