Brain Function: Novel Technologies Driving Novel Understanding - Bioinspired Approaches for Human-Centric Technologies - page 300

neurons located in layer V, but not layer II/III, was sufficient and necessary to
generate and attenuate slow oscillations, respectively. Based on patch-clamp
recordings, we proposed that the differential role of layers V and II/III in the
regulation of slow network activity is linked to the differential ability of these
neurons to propagate prolonged depolarization within and across cortical layers.
These results represent the first demonstration that the cortex is endowed with
layer-specific excitatory circuits that have distinct roles in the coordination of
ongoing cortical activity. Moreover, these findings underscore the importance of
understanding the specific functional microcircuitry of cortical layers, rather than
considering the entire cortical column as a uniform processing element.
10.4 New Optical Approaches for Imaging
and Manipulating Neuronal Activity In Vivo
with Light
Structured light or “wavefront engineering” by phase modulation is a powerful new
technique developed in the last few years (Dal Maschio et al. 2010 ). In combination
with fluorescent activity reporters, opsin-based actuators, and two-photon illumi-
nation, this technique represents a promising solution to overcome current limita-
tions of fluorescence functional imaging and optogenetics in vivo. We designed and
built a “structured light module,” a compact, simple optical path that can be easily
implemented with commercial scan heads to allow spatial shaping of laser light.
The structured light module is based on phase modulation of the light wavefront by
liquid crystal spatial light modulators (LC-SLMs). The combination of the struc-
tured light module with the scan head provides simultaneous two-photon imaging
and stimulation using two independent laser sources at different wavelengths. The
optical design allows us to combine the intrinsic three-dimensional spatial resolu-
tion of a nonlinear imaging system with simultaneous access of arbitrary regions of
the sample in time and space. We validated this approach for calcium imaging at
high frame rates (up to 70 frames/s) from multiple cells simultaneously. We also
demonstrate that this technique can be used for photo-uncaging MNI glutamate in
arbitrary 2D patterns in cultured neurons (Fig. 10.2 ).
This system can be used for simultaneous scanning imaging of Ca 2+ dyes and
holographic photostimulation of opsins, caged compounds, or photoswitchable
proteins leading to fundamental advancements in our understanding of neuronal
network function at cellular and subcellular resolution. At the same time, this
technique will allow fast, fluorescence imaging with two-photon excitation in
user-defined regions of interest and in combination with spot uncaging. These
applications, together with the observation that two-photon light penetrates deeper
in biological tissue and that some opsins can be excited with two-photon processes,
open new perspectives in the use of the present technology for in vivo studies.