Abstract
In vivo wide-field imaging of neural activity with a high spatio-temporal resolution is a challenge in modern neuroscience. Although two-photon imaging is very powerful, high-speed imaging of the activity of individual synapses is mostly limited to a field of approximately 200 µm on a side. Wide-field one-photon epifluorescence imaging can reveal neuronal activity over a field of ≥1 mm2 at a high speed, but is not able to resolve a single synapse. Here, to achieve a high spatio-temporal resolution, we combine an 8 K ultra-high-definition camera with spinning-disk one-photon confocal microscopy. This combination allowed us to image a 1 mm2 field with a pixel resolution of 0.21 µm at 60 fps. When we imaged motor cortical layer 1 in a behaving head-restrained mouse, calcium transients were detected in presynaptic boutons of thalamocortical axons sparsely labeled with GCaMP6s, although their density was lower than when two-photon imaging was used. The effects of out-of-focus fluorescence changes on calcium transients in individual boutons appeared minimal. Axonal boutons with highly correlated activity were detected over the 1 mm2 field, and were probably distributed on multiple axonal arbors originating from the same thalamic neuron. This new microscopy with an 8 K ultra-high-definition camera should serve to clarify the activity and plasticity of widely distributed cortical synapses.
Highlights
To understand neocortical information processing, it is important to be able to detect the fast events of action potentials in multiple neurons in a wide (≥1 mm) cortical area, as in addition to local cortical interactions, cortico-cortical and subcortico-cortical interactions are very important[1]
We demonstrate that spinning-disk one-photon confocal laser microscopy (SDCLM) with the 8KUHD camera (8K-SDCLM) can resolve the activity of mouse thalamocortical (TC) axonal boutons in a 1 mm[2] field of view (FOV), that it can do this at a single-bouton resolution, and that some axonal boutons at a distance of ~1 mm apart, which putatively originate from single axons, exhibit very similar activity
We demonstrated that the combination of a spinning-disk unit, a variable zoom microscope, and an 8 K ultra-high-definition (8 KUHD) camera allowed us to observe the activity of TC axonal boutons within a 1 mm[2] field in a behaving mouse
Summary
To understand neocortical information processing, it is important to be able to detect the fast events of action potentials in multiple neurons in a wide (≥1 mm) cortical area, as in addition to local cortical interactions, cortico-cortical and subcortico-cortical interactions are very important[1]. (c) Representative normalized XY (left) and XZ (right) SDCLM images of fluorescent beads with a diameter of 0.5 μm. (e) Representative normalized XZ images of fluorescent beads with a diameter of 0.5 μm (bottom) at three locations along the X axial centerline in a 1 mm[2] FOV (top). OEFM has a lower spatial resolution than TPLSM and the imaging depth within the tissue is limited to 150–200 μm[20,21], if the labeled neurons and their temporal activity are sparse, out-of-focus fluorescence changes that reflect neuronal activity do not usually cause deterioration to the changes in the fluorescence signals, and the activity of a single neuronal soma can usually be resolved in vivo[20,21,22,23]. In OEFM, light scattering within the tissue is more severe than in TPLSM, and single axonal boutons cannot be resolved
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