Fig 1.
A. Two Gabor patches will be simultaneously displayed in the left and right monocular fields of view. Mice will have to determine whether the center of mass between the two Gabor patches falls in either the left or right hemifield of view and lick towards the corresponding reward spout to receive a reward. In this figure it is assumed that both recordings and optogenetic manipulations will be performed in the left cortical hemisphere. Hence, the left visual field will be the ipsilateral one, while the right visual field will be contralateral. Gabor patches will never be displayed in the binocular field of view (red-textured area). Note that the stimulus presentation time is shorter than the onset of saccades in mice, therefore we do not expect the borders between monocular and binocular cortex to change significantly during stimulus presentation. B. Patterned illumination will be used to achieve the optogenetic inactivation of the portion of V1 that does not respond to the visual stimulus (blue areas outside the circles), as demonstrated through imaging experiments. White circles correspond to the cortical locations showing stimulus-evoked responses in both V1 and HVAs. The region of V1 being illuminated will comprise a ring of ~250 μm of tolerance, in order to account for dendritic arborizations of neurons in the non-inactivated portions of V1, and prevent the risk of reducing any input such regions might receive from surrounding regions in VI and NB conditions [60]. We will not inactivate HVAs in view of the high spatial resolution that would be required to selectively target non-responsive patches of each individual HVA.Areas P, LI, LM and AL are HVAs that are considered to be part of the mouse ventral stream. Areas RL, A, AM and PM are HVAs that are considered to be part of the mouse dorsal stream [61]. For all areas indicated in the panel, standard acronyms are used [54,61]. C. Schematic representation of the expected time course of neuronal activity during hit (solid lines) and miss (dashed lines) trials for neurons responding to the displayed Gabor patches (pink) or not (blue). Note the widespread emergence of report-related activity starting from about 200 ms after stimulus onset [31,50]. D. Timeline of the behavioral task. After a variable inter-trial interval, sensory stimuli will be displayed for 200 ms. In a fraction of trials, inactive portions of V1 will be optogenetically inactivated. Mice will be rewarded in case of a correct response, if licking is recorded during the response window, lasting from 100 ms to 1000 ms after stimulus onset.
Fig 2.
Dual-area Neuropixels 1.0 probe recordings [44] were collected during 5 recording sessions from two head-fixed mice, habituated to head-fixation and to the presentation of different visual stimuli over a period of several weeks. Additionally, during inter-trial periods mice were exposed to a gray background, to which they became habituated. Total number of identified single neurons following spike sorting with Kilosort 2.0 [56]: 137 neurons in V1 and 95 neurons in the anterior cingulate cortex (ACC). Mice expressed the inhibitory opsin iC++ [62] in pyramidal neurons in ACC. This was achieved via viral injection of an AAV vector mediating the expression of iC++ under the CaMKII promoter. A. The activity of V1 units was quantified as a function of which visual background was presented (black screen, grey screen or static white noise pattern – grey-valued bins – shown for periods of several minutes). Recordings were subdivided into non-overlapping windows of 200 ms, during which we computed the average firing rates of detected neurons (left), and the fraction of neurons not displaying any action potential during each 200 ms window (right). We found that, only when displaying a grey visual background activity in V1 was not significantly higher than 0 for both metrics. Asterisks indicate values significantly different from either 0 (firing rates) or 1 (fraction of inactive neurons) (p < 0.05, one-sided t-test). B. The effect of optogenetic inactivation of V1 (over 200 ms windows) was compared for both V1 and ACC as a function of visual background. Optogenetic inactivation of V1 was effective in reducing V1 activity across all visual backgrounds. However, a signification reduction was observed in ACC, a downstream area to V1, only when displaying a static white noise. Asterisks indicate significant differences between baseline firing rates and epochs with optogenetic illumination (p < 0.05, paired t-test); note that a smaller dataset was used in panel B compared to panel A. Taken together, these results indicate that the use of a grey background induces a level of spontaneous activity in V1 compatible with the MA level, i.e., statistically indistinguishable from 0; the use of a black background instead elicits some spontaneous activity, but at a level that is not high enough to make a difference to downstream areas – and thus in line with the definition of the VI level. Finally, the use of a random pattern as a visual background further increases spontaneous V1 activity to a level compatible with the NB level.
Fig 3.
Cranial window and optogenetic inactivation.
A. Example of PDMS window placed on the left hemisphere. Dental cement (grey) is used to stably fix the window onto the remaining skull. B. Example histological sections (top: 10X magnification; bottom: 5X magnification, coronal section located 4.20 mm posterior to bregma) showing the expression of the red-shifted inhibitory opsin Jaws in the visual cortex of a pilot animal. In the final experiment we will perform multiple viral injections in GCaMP-expressing mice to cover the entirety of V1. C. Example neuron showing Jaws-mediated inactivation upon illumination with a 637 nm laser (shaded area). Top: raster plot. Bottom: Average peri-event time histogram (PETH) ± SEM.
Fig 4.
Patterned inactivation protocol.
A. Optical path of the setup for simultaneous widefield calcium imaging (including monitoring of hemodynamic responses) and patterned illumination via a laser beam projector. Violet: LED light used to monitor the hemodynamic response (red path). Blue: LED light used to induce GCaMP6f fluorescence (green path). B. Widefield calcium imaging will be used to identify the portion of the visual cortex responding to Gabor patches presented in the experimentally relevant locations (Fig 5B). Left: widefield calcium response to a single Gabor patch, after correction for hemodynamic responses [43]. Right: outline of the visually responsive portion of V1 is computed and overlaid on the vasculature map for localizing it. C. Top-left: A sign map [42] is computed to identify the extension of V1 (data and code obtained from https://github.com/zhuangjun1981/NeuroAnalysisTools). Top-right: The outline of V1, as identified by the sign map, is overlaid on the vasculature map. Bottom-left: Overlay of the outline of V1 and of the portion of V1 responsive to the presented Gabor patch (see panel B). Bottom-right: The mask for patterned illumination is computed as the negative image of the portion of V1 responding to a Gabor patch. Red light is used to activate the inhibitory opsin Jaws. D. The final illumination pattern (see panel C) is projected on the cortex for inactivation the portion of V1 not responding to the presented Gabor patch.
Fig 5.
A. Various configurations of locations of the Gabor patches will be presented and coupled with optogenetic inactivation. Bilateral configurations will be used to answer to test the theories’ predictions (cf. Fig 6). In order to make the experiment feasible, we have selected a set of configurations for which the theories under scrutiny yield the most distinct predictions (cf. Fig 6). Importantly, some additional configurations (shaded in blue) are necessary to maintain a proper balance between stimuli rewarded with left vs. right licks – to prevent the insurgence of a response bias to either side. Two bilateral configurations will be coupled with optogenetic inactivation of background activity (here indicated with a striped shading). Probe trials will be used to estimate false alarm rates and any effect of optogenetic illumination on it. Finally, unilateral configurations will be used to test if optogenetic inactivation induces any bias in response side, in the absence of a stimulus being presented in the inactivated hemifield of view. Fractions and number of trials for each category are only an estimate that will depend on animals’ performance and on the actual number of trials in each experimental session. B. Diagram indicating the positions of the Gabor patches that will be used in the actual experimental sessions. Compared to the outline shown in Fig 1, we will have to adjust the locations where the Gabor patches are shown based on the actual coverage of the visual field by the screen where visual stimuli are displayed within the experimental setup. Specifically, while the visual field extends up to 140 deg beyond the midline, the screens only reach 110 deg. This introduces a possible confounding factor. Specifically, the line splitting each hemifield of view in half (dashed line) does not overlap with the line splitting the portion of the screen visible within each hemifield of view in two (dotted line). Under the assumption that optogenetic inactivation will induce a hemineglect-like deficit, we cannot predict if mice will respond based on the position of the still-perceived Gabor patch with respect to either the dashed or dotted line, i.e., based on the position of the Gabor on the screen or on the field of view. For this reason, to avoid possible confounding factors, we will avoid displaying Gabor patches in the portion of the visual field (shaded in blue) between these two lines, where no prediction can be made about the behavioral responses of mice.
Fig 6.
Scheme of experimental predictions.
Various configuration of locations of the Gabor patches yield distinct experimental predictions, based on the level of background activity, and on whether inactivation of inactive neurons in V1 and HVAs in one hemisphere will induce hemineglect. Specifically, different predictions can be made based on whether the location of the center of mass (CM) for all Gabor patches falls in the contralateral or ipsilateral hemifield of view (with respect to the inactivated hemisphere), as well as on the position of the CM for the patches shown in the ipsilateral hemifield (i.e., based on whether this falls in the contralateral or ipsilateral half-hemifield - see also Fig 1A). Overall, four different configurations of stimuli will be tested (four panels in the figure). The same graphic conventions are used as in Figs 1 and 5. The area in the right hemifield with a striped background indicates the region of the visual field corresponding to the optogenetically manipulated portion of V1. Note that the regions where Gabor patches are shown correspond to portions of V1 that will not be optogenetically inactivated (see also Fig 1B). In this figure we assume that the left hemisphere is the inactivated one (hence: right is contralateral and left is ipsilateral). The “Control” label below each table indicates the expected behavior in the absence of optogenetic inactivation. MA: minimally active; VI: virtually inactive; NB: normal background; CM: center of mass; orange triangle: location of the CM for all shown patches; green: location of the phenomenologically experienced CM if optogenetic inactivation leads to functional hemineglect of the contralateral hemifield of view. The consequence of functional hemineglect (as predicted by either IIT or PP-NREP) is that mice will not experience visual space in the hemifield contralateral to the optogenetically inactivated hemisphere. Thus, the residual hemifield will be split into two half-hemifields, which will subjectively be experienced as the left and right hemifields. Therefore, in the presence of functional hemineglect, mice are expected to respond not based on the physical location of the center of mass but based on whether the ipsilaterally-presented Gabor patch (overlapping with the phenomenogically experienced CM) falls in either the left or right half-hemifield. Basically, IIT and NREP predict that, under some circumstances (different between IIT and NREP) that the total experienced space will shrink relative to the absolute space of the external visual field, and that mice will only respond based on the perceived location of the ipsilateral Gabor patch. Note that not all configurations are equally informative. For example, the predictions of IIT and PP-NREP for configuration 4 are similar and do not generally predict a major effect of optogenetic inactivation. In contrast, all theories have very different predictions on configuration 3. Therefore, only the most informative configurations will be used in the final experiments (cf. Fig 5A). Finally, while predictions are different for configuration 2, this configuration entails showing Gabor patches very close to the midline. We expect this configuration to be difficult for animals, hence we will not include it in the final experiment (cf. Fig 5A).
Table 1.
Scheme of experimental predictions related to configuration 3 (see also Fig 6).