Two-photon imaging of activity evoked in populations of neurons by simple visual stimuli in the primary visual cortex of awake rats

Two-photon imaging of the activity of populations of neurons allows detailed investigation of potential population coding strategies used by networks of cortical neurons to represent sensory stimuli and how individual neurons may contribute to this representation. Recent studies from several groups have used this approach to examine evoked responses in populations of neurons in primary sensory areas in anaesthetized animals. We have extended these investigations here to examine the representation of simple visual stimuli in populations of superficial neurons in the primary visual cortex of awake rats. Rats were trained to tolerate sessions of headfixation of sufficient length to allow loading of neurons with the Ca2+-sensitive indicator OGB-1 and subsequent recording of responses to basic visual stimuli (drifting gratings and moving lines). Bolus loading of the OGB-1 was targeted to the cortical area representing the visual space around the vertical meridian and directly in front of the animals nose using optical intrinsic signal imaging and was confirmed subsequently via receptive field mapping of the loaded area using local field potential recording through the loading pipette. Movements of the eyes were monitored using an infra-red camera mounted close to each eye. Drifting grating stimuli of different angles evoked strong activity in discrete subsets of neurons which were scattered in the field of view (ie not organized into specific areas for different angles), as described previously for anaesthetized rodents. Movements of the eyes were not systematically observed during the experiments, though rapid shifts of the position of the pupil, usually in the rostral-caudal plane, were occasionally observed, particularly if the animal was startled. We also found no evidence for systematic movements of the pupil to follow a moving object, such as a line or bar. The experimental setup also allowed for the animal to be anaesthetized during the recording, thus allowing direct comparison of the stimulus responses of the same neurons with the animal under anaesthesia. Additionally, the setup allowed simultaneous whole-cell or cell-attached electrophysiological recordings that could be used to assess the sensitivity of the Ca2+-imaging data for detecting individual action potentials. Using this approach, we aim to provide further insights into coding strategies employed by the visual cortex in awake rats and to what extent these strategies can be inferred from recordings made in anaesthetized animals