Abstract
SummarySensory information is encoded within the brain in distributed spatiotemporal patterns of neuronal activity. Understanding how these patterns influence behavior requires a method to measure and to bidirectionally perturb with high spatial resolution the activity of the multiple neuronal cell types engaged in sensory processing. Here, we combined two-photon holography to stimulate neurons expressing blue light-sensitive opsins (ChR2 and GtACR2) with two-photon imaging of the red-shifted indicator jRCaMP1a in the mouse neocortex in vivo. We demonstrate efficient control of neural excitability across cell types and layers with holographic stimulation and improved spatial resolution by opsin somatic targeting. Moreover, we performed simultaneous two-photon imaging of jRCaMP1a and bidirectional two-photon manipulation of cellular activity with negligible effect of the imaging beam on opsin excitation. This all-optical approach represents a powerful tool to causally dissect how activity patterns in specified ensembles of neurons determine brain function and animal behavior.
Highlights
Within brain circuits, information about sensory stimuli is encoded in complex spatial and temporal patterns of activity distributed across cells (Kampa et al, 2011; Ohki et al, 2005; Sawinski et al, 2009)
To test whether using a blue light-sensitive channelrhodopsin would lead to a reduction in the undesired cross-activation during activity reporter imaging, we expressed ChR2 and C1V1(T/T) in cultured hippocampal neurons and characterized the photocurrent evoked by two-photon scanning at the wavelengths typically used for calcium imaging of green and red calcium indicators (920 and 1,080 nm, respectively) (Figure S1)
High Spatial Resolution Two-Photon Holographic Stimulation In Vivo To stimulate neurons with high spatial resolution in vivo, we used a liquid crystal spatial light modulator (SLM)-based holographic module, which was integrated in a commercial laser scanning two-photon microscope (Figure 1A) (Dal Maschio et al, 2010, 2011), and we programmed the holographic module to project on the sample plane elliptical shapes that were centered on the cell body of target neurons (Figure 1B; Figure S2)
Summary
Information about sensory stimuli is encoded in complex spatial and temporal patterns of activity distributed across cells (Kampa et al, 2011; Ohki et al, 2005; Sawinski et al, 2009). Using statistical analysis and correlative evidence to causally test which sensory features are encoded in neural circuits and how this information is used to drive behavior may prove difficult (Panzeri et al, 2017) To achieve this goal, we would ideally need a method to monitor and bidirectionally perturb the activity of multiple neurons maintaining single-cell resolution. This shoulder toward shorter wavelengths (Yizhar et al, 2011) typical for red-shifted opsins
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