In vivo two-photon voltage-sensitive dye imaging reveals top-down control of cortical layers 1 and 2 during wakefulness

Abstract

Conventional methods of imaging membrane potential changes have limited spatial resolution, particularly along the axis perpendicular to the cortical surface. The laminar organization of the cortex suggests, however, that the distribution of activity in depth is not uniform. We developed a technique to resolve network activity of different cortical layers in vivo using two-photon microscopy of the voltage-sensitive dye (VSD) ANNINE-6. We imaged spontaneous voltage changes in the barrel field of the somatosensory cortex of head-restrained mice and analyzed their spatiotemporal correlations during anesthesia and wakefulness. EEG recordings always correlated more strongly with VSD signals in layer (L) 2 than in L1. Nearby (<200 mum) cortical areas were correlated with one another during anesthesia. Waking the mouse strongly desynchronized neighboring cortical areas in L1 in the 4- to 10-Hz frequency band. Wakefulness also slightly increased synchrony of neighboring territories in L2 in the 0.5- to 4.0-Hz range. Our observations are consistent with the idea that, in the awake animal, long-range inputs to L1 of the sensory cortex from various cortical and thalamic areas exert top-down control on sensory processing.

Fig. 1.

VSD responses to sensory stimulation can be imaged by two-photon microscopy. (a) Combined in vivo setup for intrinsic and two-photon imaging. (b) Superposition of a reflected-light image of the surface vasculature and barrel positions (blue, C1; magenta, C2; green, C3). Colored areas show where reflectivity changes were >0.1% in response to whisker deflection. White line, line scan position and length during two-photon imaging. (c) Averaged (n = 400 trials) dye responses to whisker stimulation for focal depths of 40, 200, and 400 μm at an excitation wavelength of 1,020 nm. Dashed line, onset of stimulation of C2, the principal whisker for the line scan position. Red, fits of α function. (d–f) VSD signal amplitude in units of ΔF/F (d), latency (e), and decay (f) as a function of depth (n = 4 mice). (g) VSD signal for 960- and 1,020-nm excitation at a depth of 80 μm under identical stimulus conditions (200 trials).

Fig. 2.

Imaging and time-domain correlation analysis of spontaneous activity. (a) Two-photon images at depths of 40 μm (Left) and 160 μm (Right) below the pia. Lines across image centers, line scan segments used for calculation of CC and coherence. (b–d) Data from one animal. (e and f) Pooled data (n = 9 mice). (b) EEG (Upper) and simultaneously measured VSD signals (Lower) show corresponding activity. Orange trace, average over the pixels of the orange segment in a; black trace, 200-ms boxcar filtered. (c) CC of total VSD signal and EEG. (d) CC of adjacent 64-μm-wide segments of the VSD signals averaged over all pairs. (e and f) Same as in c and d, except pooled over all mice.

Fig. 3.

Frequency domain analysis. Average (n = 9) power and coherence spectra (magnitude) of EEG and VSD for two depths (L1, 40 μm; L2, 160 μm) and for different arousal states. (a and b) Power spectra of the EEG (a) and VSD (b) for anesthetized (blue) and awake (red) states. (c and d) Coherence was either between the EEG and the VSD signal for the entire line scan length (c) or between adjacent 64-μm-wide line segments (d). Power and coherence spectra were smoothed with a 0.25-Hz boxcar. (e) Coherence within specific bands (either 0.5–4 Hz or 4–10 Hz) as a function of depth. (f) Coherence as a function of distance.

Fig. 4.

AMPA/kainate receptors are required for coherence to be state-dependent. Ratio of coherence during wakefulness to coherence during anesthesia before CNQX (black), during CNQX (red), and after washout (blue) (n = 3).