Investigating second messenger signaling in vivo.

All known second messengers are small molecules without complex structural features. However, each of them can mediate different, very specific cellular responses upon arrival of distinct extracellular cues in one and the same cell. From this follows the question of how signal specificity is achieved in space and time. Recent work showed that three factors play major roles in determining signal specificity: intensity, pattern, and compartmentalization of signals. Thus, for understanding the signaling in any specific context, generic information on the involvement of second messenger pathway(s) is insufficient and only the precise, time- and space-resolved monitoring of these processes will lead to meaningful insights. Even more demanding, many second messenger-based signaling events can only occur using tissue-specific morphological arrangements (e.g., striated organization of muscle and synaptic specializations in neurons), making the visualization of intact tissue mandatory. Finally, to appreciate the physiological relevance of second messenger signals, multiparametric readouts are generally needed. This chapter illustrates modes of multiparametric analysis of signaling in live mouse skeletal muscle and briefly discusses possible applications of the technique in other tissues including heart and brain.