Voltage imaging of cortical oscillations in layer 1 with two-photon microscopy

Abstract

Membrane voltage oscillations in layer 1 (L1) of primary sensory cortices might be important indicators of cortical gain control, attentional focusing, and signal integration. However, electric field recordings are hampered by the low seal resistance of electrodes close to the brain surface. To study L1 membrane voltage oscillations, we synthesized a new voltage-sensitive dye, di1-ANNINE-6plus, that can diffuse into tissue. We applied it with a new surgery, leaving the dura intact but allowing injection of large quantities of staining solution, and imaged cortical membrane potential oscillations with two-photon microscopy depth-resolved (25 to 100 µm below dura) in anesthetized and awake mice. We found delta (0.5-4 Hz), theta (4-10 Hz), low beta (10-20 Hz), and low gamma (30-40 Hz) oscillations. All oscillations were stronger in awake animals. While the power of delta, theta, and low beta oscillations increased with depth, the power of low gamma was more constant throughout L1. These findings identify L1 as an important coordination hub for the dynamic binding process of neurons mediated by oscillations.

Significance Statement Here, we describe a new voltage-sensitive dye, surgery technique, and voltage measurements in cortical L1. The new voltage-sensitive dye di1-ANNINE-6plus shows the same high sensitivity as previous ANNINE-6 dyes but diffuses better in tissue. The bubble surgery allows to load up to 10 microliters of dye or drug solution between dura and brain without dura removal or disturbance of cortical L1 in the mouse. Voltage imaging with di1-ANNINE-6plus and two-photon microscopy allows to measure membrane voltage spectra in L1 depth resolved up to a frequency of 50 Hz in 30-second episodes and 500-femtoliter (=1x5x100 µm³ line-scan volume) tissue volumes. Our measurements reveal oscillations below 20 Hz and slow gamma oscillations in the range of 35Hz in L1 of behaving mice.