205 Chapter 11 In Vivo Ca 2+ Imaging of the Living Brain Using Multi-cell Bolus Loading Technique Gerhard Eichhoff, Yury Kovalchuk, Zsuzsanna Varga, Alexei Verkhratsky, and Olga Garaschuk Abstract This chapter overviews the use of acetoxymethyl (AM) ester-based multi cell bolus loading (MCBL) tech- nique for in vivo Ca 2+ imaging of neural circuits. This technique provides anatomically targeted, rapid and non-invasive staining of both neurones and glia with small molecule Ca 2+ indicators thus allowing real- time imaging of brain activity. We describe the protocols for staining cells in newborn, juvenile, adult and aged tissue; discuss critical steps and possible pitfalls and introduce the multicolor imaging approach for functional characterization of specific cell types. We show, furthermore, that the use of MCBL can be extended to label other elements within the brain tissue as, for example, amyloid plaques in a mouse model of Alzheimer’s disease (AD). Finally, we illustrate the use of MCBL for two-photon calcium imaging of neurones and glia in the aged mouse cortex as well as the mouse cortex in an animal model of AD. Key words: In vivo two-photon microscopy, Calcium indicator dye, Staining technique, Calcium imaging, Ageing, Alzheimer’s disease Monitoring physiological activity of neural circuits is fundamental for understanding of the brain function. Mammalian brain is the extremely complex and dense cellular network. Its grey matter is composed of two closely associated cellular circuits, the neuronal net and the glial web. Closely apposed neuronal and glial membranes compartmentalize the extracellular space into narrow subregions and clefts, where neurotransmitters, neuromodulators and hormones diffuse and undergo complex processes of enzy- matic degradation and active uptake. The three-dimensional organization of the brain is therefore critically important for the integration within neural circuits, and the function of such 1. Introduction A. Verkhratsky and Ole H. Petersen (eds.), Calcium Measurement Methods, Neuromethods, vol. 43 DOI 10.1007/978-1-60761-476-0_11, © Humana Press, a part of Springer Science + Business Media, LLC 2010