Optical Imaging of Brain Activity In Vivo Using Genetically Encoded Probes

Genetically encoded reporters of neural activity have great promise as tools for imaging brain function in vivo. Reproducible labeling of specific cell types and the continuous presence of indicators for long-term experiments are the most prominent advantages of this new methodology. It is becoming increasingly acknowledged that genetically encoded optical reporters, combined with advanced methods for recording these optical signals, will be the tool of choice for monitoring neural activity in the intact animal. Since the appearance of groundbreaking and influential descriptions of genetically encodable reporters of neuronal activity [1–4] intense efforts have been made to apply these new probes under in vivo conditions. To date, however, a relatively small number of optical reporters have proven useful for in vivo brain imaging or even in vitro neural preparations, and only a subset of these have proven useful for imaging in the intact mammalian brain. The amount of time that it has taken for these tools to mature is due to serious technical challenges in developing optical reporters that generate a sufficient signal in the intact brain, function correctly at mammalian body temperature, and throughout the life of the neuron are expressed at sufficient levels in the desired neurons without significantly altering their function. Nonetheless, many of these hurdles have been overcome in recent years, and several classes of genetically encoded activity reporters work robustly when expressed in a variety of different systems. Continued effort in developing probe and cell type–specific expression systems also promises to increase the utility and number of these reporters in the very near future. In theory, the use of genetically encoded optical reporters of neural activity in vivo should be similar to (or less demanding than) that of classical, synthetic optical indicators. Indeed, like their synthetic counterparts, the major classes of genetically encoded reporters sense calcium or voltage, with a third class (the pHluorins) reporting synaptic vesicle cycling. Because genetically encoded reporters are typically derivatives of naturally expressed proteins such as calmodulin or ion channel subunits, the potential for interaction with endogenous proteins and enzyme substrates adds another level of complexity to experimental design and data interpretation. Thus, while many of the same technical issues seen with synthetic optical reporters apply to the use of genetically encoded probes, additional factors dependent on the specific design and mechanism of each type of indicator must also be considered when using these probes to measure and interpret neural activity in vivo. In this chapter, we will review the design principles underlying three major classes of genetically encoded indicators—calcium sensors, reporters of transmitter release, and voltage sensors—as well as strategies for expressing these indicators in vivo. We will focus on the use of these probes in the mammalian brain, where their implementation has been the most challenging, although work in nonmammalian systems (i.e., zebrafish, Drosophila) and in in vitro preparations will be discussed in cases where this work has yielded important insights. We will then present examples using two of these sensors—synaptopHluorin (spH) and GCaMP2—to monitor sensory coding and postsynaptic processing in the mouse olfactory bulb. GCaMP2 and spH work via very different mechanisms and report distinct—though related—aspects of neural activity; consequently, each probe presents different advantages and difficulties when monitoring brain function in vivo. While the work presented here has been done primarily in the olfactory system, each of these probes has proven robust in its ability to report activity in a variety of neuronal systems, and so the principles discussed in this chapter should be generally applicable to imaging elsewhere in the brain.