The cerebral cortex is organized in vertical columns
that contain neurons with similar functions. The
cellular micro-architecture of such columns is an
essential determinant of brain dynamics and cortical
information processing. However, a detailed understanding
of columns is incomplete, even in the best
studied cortical regions, and mostly restricted to
the upper cortical layers. Here, we developed a
two-photon Ca2+-imaging-based method for the serial
functional mapping of all pyramidal layers of the
mouse primary auditory cortex at single-neuron resolution
in individual animals. We demonstrate that
the best frequency-responsive neurons are organized
in all-layers-crossing narrow columns, with
fuzzy boundaries and a bandwidth of about one
octave. This micro-architecture is, in many ways,
different from what has been reported before, indicating
the region and stimulus specificity of functional
cortical columns in vivo.