  The glow of a starry night sky, the smell of a freshly brewed cup of coffee
or the sound of ocean waves breaking on the beach are representations of the
physical world that have been created by the dynamic interactions of thousands
of neurons in our brains. How the brain mediates perceptions, creates thoughts,
stores memories and initiates actions remains one of the most profound puzzles
in biology, if not all of science. A key to a mechanistic understanding of how
the nervous system works is the ability to analyze the dynamics of neuronal
networks in the living organism in the context of sensory stimulation and
behaviour. Dynamic brain properties have been fairly well characterized on the
microscopic level of individual neurons and on the macroscopic level of whole
brain areas largely with the help of various electrophysiological techniques.
However, our understanding of the mesoscopic level comprising local populations
of hundreds to thousands of neurons (so called 'microcircuits') remains
comparably poor. In large parts, this has been due to the technical
difficulties involved in recording from large networks of neurons with
single-cell spatial resolution and near- millisecond temporal resolution in the
brain of living animals. In recent years, two-photon microscopy has emerged as
a technique which meets many of these requirements and thus has become the
method of choice for the interrogation of local neural circuits. Here, we
review the state-of-research in the field of two-photon imaging of neuronal
populations, covering the topics of microscope technology, suitable fluorescent
indicator dyes, staining techniques, and in particular analysis techniques for
extracting relevant information from the fluorescence data. We expect that
functional analysis of neural networks using two-photon imaging will help to
decipher fundamental operational principles of neural microcircuits.
