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Abstract

While the neuronal basis of certain behaviors and sensory modalities can be studied under anesthesia or head-fixation, the full gamut of neural activity and its functions is accessible only in awake and unrestrained animals. We therefore developed a miniaturized, head mounted 2-photon microscope or “fiberscope” capable of resolving functional action-potential derived fluorescent signals from individual cortical cells. Using the fiberscope, we imaged spontaneous and stimulus-evoked activity from populations of layer 2/3 neurons and astrocytes in the visual cortex in both anesthetized and freely moving rats. The fiberscope weighs 5.5 g, and employs a custom-designed water immersion lens and a non-resonant fiber scanner leveraged by a piezo-element. Excitation light is delivered to the mobile animal through a single-mode optical fiber. Emitted light is collected through a plastic optical fiber before being split into a green channel, for the calcium-indicator Oregon Green BAPTA-1 (OGB1), and a red channel for the specific astrocyte marker sulforhodamin-101. Images acquired through the fiberscope were stable through a range of behaviors, even when the animal is moving vigorously (> 0.6 m/s). Using optical intrinsic imaging to locate the visual cortex binocular region, OGB1 was bulk loaded into cells within this region and remained viable over the course of the four hour experiments. Movement-based artifacts were small and could be successfully corrected offline using custom built software. Fiberscope imaging of neurons under anesthesia revealed robust stimulus responses and orientation selectivity. For imaging in freely-moving animals, the rat was allowed to run in a rectangular arena with one wall made of transparent Perspex behind which static visual stimuli were presented on a CRT monitor. Imaging sessions were run in darkness and recorded using infra-red videography. Transients could be observed in a subgroup of cells in response to activation of the visual stimulus. Further, movements of the animal which resulted in the stimulus sweeping through the animals visual field also evoked transients in a subgroup of the imaged cells. Thus, the miniaturized two-photon fiberscope can record spontaneous and stimulus-evoked Ca2+ transients in freely moving animals. We expect that the fiberscope will facilitate the study of neuronal population activity of during complex behavioral tasks.