Animals move their head and eyes as they explore and sample the visual scene. Previous studies have demonstrated neural correlates of head and eye movements in rodent primary visual cortex (V1), but the sources and computational roles of these signals are unclear. We addressed this by combining measurement of head and eye movements with high density neural recordings in freely moving mice. V1 neurons responded primarily to gaze shifts, where head movements are accompanied by saccadic eye movements, rather than to head movements where compensatory eye movements stabilize gaze. A variety of activity patterns immediately followed gaze shifts, including units with positive, biphasic, or negative responses, and together these responses formed a temporal sequence following the gaze shift. These responses were greatly diminished in the dark for the vast majority of units, replaced by a uniform suppression of activity, and were similar to those evoked by sequentially flashed stimuli in head-fixed conditions, suggesting that gaze shift transients represent the temporal response to the rapid onset of new visual input. Notably, neurons responded in a sequence that matches their spatial frequency preference, from low to high spatial frequency tuning, consistent with coarse-to-fine processing of the visual scene following each gaze shift. Recordings in foveal V1 of freely gazing head-fixed marmosets revealed a similar sequence of temporal response following a saccade, as well as the progression of spatial frequency tuning. Together, our results demonstrate that active vision in both mice and marmosets consists of a dynamic temporal sequence of neural activity associated with visual sampling.

Mouse data :

Mice were initially implanted with a titanium headplate over primary visual cortex to allow for head-fixation and attachment of head-mounted experimental hardware. After three days of recovery, widefield imaging53 was performed to help target the electrophysiology implant to the approximate center of left monocular V1. A miniature connector (Mill-Max 853-93-100-10-001000) was secured to the headplate to allow repeated, reversible attachment of a camera arm, eye/world cameras and IMU21,22. In order to simulate the weight of the real electrophysiology drive for habituation, a ‘dummy’ electrophysiology drive was glued to the headplate. Animals were handled by the experimenter for several days before surgical procedures, and subsequently habituated (~45 min total) to the spherical treadmill and freely moving arena with hardware tethering attached for several days before experiments. 

The electrophysiology implant was performed once animals moved comfortably in the arena. A craniotomy was performed over V1, and a linear silicon probe (64 or 128 channels, Diagnostic Biochips P64-3 or P128-6) mounted in a custom 3D-printed drive (Yuta Senzai, UCSF) was lowered into the brain using a stereotax to an approximate tip depth of 750 µm from the pial surface. The surface of the craniotomy was coated in artificial dura (Dow DOWSIL 3-4680) and the drive was secured to the headplate using light-curable dental acrylic (Unifast LC). A second craniotomy was performed above left frontal cortex, and a reference wire was inserted into the brain. The opening was coated with a small amount of sterile ophthalmic ointment before the wire was glued in place with cyanoacrylate. Animals recovered overnight and experiments began the following day.

The camera arm was oriented approximately 90 deg to the right of the nose and included an eye-facing camera (iSecurity101 1000TVL NTSC, 30 fps interlaced), an infrared-LED to illuminate the eye (Chanzon, 3 mm diameter, 940 nm wavelength), a wide-angle camera oriented toward the mouse’s point of view (BETAFPV C01, 30 fps interlaced) and an inertial measurement unit acquiring three-axis gyroscope and accelerometer signals (Rosco Technologies; acquired 30 kHz, downsampled to 300 Hz and interpolated to camera data). Fine gauge wire (Cooner, 36 AWG, #CZ1174CLR) connected the IMU to its acquisition box, and each of the cameras to a USB video capture device (Pinnacle Dazzle or StarTech USB3HDCAP). A top-down camera (FLIR Blackfly USB3, 60 fps) recorded the mouse in the arena.

The electrophysiology headstage (built into the silicon probe package) was connected to an Open Ephys acquisition system via an ultra thin cable (Intan #C3216). Electrophysiology data were acquired at 30 kHz and bandpass filtered between 0.01 Hz and 7.5 kHz. We first used the Open Ephys GUI (https://github.com/open-ephys/plugin-GUI) to assess the quality of the electrophysiology data, then recordings were performed in Bonsai54 using custom workflows (https://github.com/nielllab/FreelyMovingEphys). System timestamps were collected for all hardware devices and later used to align data streams through interpolation.

Marmoset data:

Electrophysiological recordings were performed using 2x32 channel silicon electrode arrays (http://www.neuronexus.com). Probes included 2 sharpened tip shanks of 50µm width spaced 200 µm apart, each containing 32 channels separated by 35 µm. In one animal we used a semi-chronic microdrive (EDDS Microdrive system, https://microprobes.com) to place electrodes in cortex for 1-2 weeks over which we made 3-6 recordings. In the second animal we used a custom micro-drive (https://marmolab.bcs.rochester.edu/resources/) to place and remove electrodes daily. Arrays were lowered slowly through silastic into cortex using a thumb screw.

Data were amplified and digitized at 30 kHz with Intan headstages (Intan) using the Open Ephys GUI (https://github.com/open-ephys/plugin-GUI). The wideband signal was high-pass filtered by the headstage at 0.1 Hz, preprocessed by common-average referencing across all channels, and then high-pass filtered at 300 Hz. The resulting traces were spike sorted using Kilosort2. Outputs from the spike sorting algorithms were manually labeled using ’phy’ GUI (https://github.com/kwikteam/phy). Any units that were either physiologically implausible based on the lack of a waveform with a trough followed by a peak or with an inter-spike interval (ISI) distribution with more than 1% of the spikes under 1 ms were excluded from analyses.

Gaze position was monitored using infra-red eye tracking methods described previously63. Briefly, the 1st and 4th Purkinje images (P1 and P4) were visualized using a collimated IR light source and tracked at 593 frames per second to estimate the 2D eye angle. The eye tracker was manually calibrated to adjust the offset and gain (horizontal and vertical) by showing marmoset monkeys small windowed face images at different screen positions to obtain their fixation as described previously60,61. Saccadic eye movements were identified automatically using a combination of velocity and acceleration thresholds64.