<i>In vivo</i> two-photon calcium imaging of neuronal networks

TLDR

Two‑photon calcium imaging enables in vivo monitoring of individual neuronal activity, yet until now has been restricted to dye‑loaded single neurons, with membrane‑permeant dyes only effective in cultured or immature slice preparations. The authors present a versatile method for loading membrane‑permeant fluorescent calcium indicators into large populations of neurons. They developed a pressure‑ejection local dye‑delivery protocol that works with dyes such as Ca‑green‑1 AM, Fura‑2 AM, Fluo‑4 AM, and Indo‑1 AM, and successfully applied it to mouse brain tissue from newborn to adult in vivo and in vitro. Using this technique, two‑photon Ca²⁺ imaging through the intact skull revealed whisker‑evoked calcium transients in a subset of layer 2/3 barrel‑cortex neurons, demonstrating the method’s suitability for real‑time, single‑cell resolution analysis of intact neuronal circuits.

Abstract

Two-photon calcium imaging is a powerful means for monitoring the activity of distinct neurons in brain tissue in vivo . In the mammalian brain, such imaging studies have been restricted largely to calcium recordings from neurons that were individually dye-loaded through microelectrodes. Previous attempts to use membrane-permeant forms of fluorometric calcium indicators to load populations of neurons have yielded satisfactory results only in cell cultures or in slices of immature brain tissue. Here we introduce a versatile approach for loading membrane-permeant fluorescent indicator dyes in large populations of cells. We established a pressure ejection-based local dye delivery protocol that can be used for a large spectrum of membrane-permeant indicator dyes, including calcium green-1 acetoxymethyl (AM) ester, Fura-2 AM, Fluo-4 AM, and Indo-1 AM. We applied this dye-loading protocol successfully in mouse brain tissue at any developmental stage from newborn to adult in vivo and in vitro. In vivo two-photon Ca 2+ recordings, obtained by imaging through the intact skull, indicated that whisker deflection-evoked Ca 2+ transients occur in a subset of layer 2/3 neurons of the barrel cortex. Thus, our results demonstrate the suitability of this technique for real-time analyses of intact neuronal circuits with the resolution of individual cells.