Neural activity imaging with genetically encoded calcium indicators

Abstract

Genetically encoded calcium indicators (GECIs), together with modern microscopy, allow repeated activity measurement, in real time and with cellular resolution, of defined cellular populations. Recent efforts in protein engineering have yielded several high-quality GECIs that facilitate new applications in neuroscience. Here, we summarize recent progress in GECI design, optimization, and characterization, and provide guidelines for selecting the appropriate GECI for a given biological application. We focus on the unique challenges associated with imaging in behaving animals.

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, [Ca^2 +], 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 Ca^2 + 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.