A Comparison of Visual Response Properties in the Lateral Geniculate Nucleus and Primary Visual Cortex of Awake and Anesthetized Mice

Abstract

The cerebral cortex of the mouse has become one of the most important systems for studying information processing and the neural correlates of behavior. Multiple studies have examined the first stages of visual cortical processing: primary visual cortex (V1) and its thalamic inputs from the dorsal lateral geniculate nucleus (dLGN), but more rarely in the lateral posterior nucleus (LP) in mice. Multiple single-unit surveys of dLGN and V1, both with electrophysiology and two-photon calcium imaging, have described receptive fields in anesthetized animals. Increasingly, awake animals are being used in physiological studies, so it is important to compare neuronal responses between awake and anesthetized state. We have performed a comprehensive survey of spatial and temporal response properties in V1, dLGN, and lateral posterior nucleus of both anesthetized and awake animals, using a common set of stimuli: drifting sine-wave gratings spanning a broad range of spatial and temporal parameters, and sparse noise stimuli consisting of flashed light and dark squares. Most qualitative receptive field parameters were found to be unchanged between the two states, such as most aspects of spatial processing, but there were significant differences in several parameters, most notably in temporal processing. Compared with anesthetized animals, the temporal frequency that evoked the peak response was shifted toward higher values in the dLGN of awake mice and responses were more sustained. Further, the peak response to a flashed stimulus was earlier in all three areas. Overall, however, receptive field properties in the anesthetized animal remain a good model for those in the awake animal.

Significance statement: The primary visual cortex (V1) of the mouse and its inputs from visual thalamus (dLGN), have become a dominant model for studying information processing in the brain. Early surveys of visual response properties (receptive fields) were performed in anesthetized animals. Although most recent studies of V1 have been performed in awake animals to examine links between vision and behavior, there have been few comprehensive studies of receptive field properties in the awake mouse, especially in dLGN and lateral posterior nucleus. We have performed a comparative survey of receptive fields in dLGN, lateral posterior nucleus, and V1 in anesthetized and awake mice. We found multiple differences in processing of time-varying stimuli, whereas the spatial aspects of receptive fields remain comparatively unchanged.

Keywords: LGN; LP; anesthesia; mouse; visual cortex.

Figure 1.

Localization of recordings. A, Schematic of a Neuronexus Edge 32 probe (32 channels on one side of a single shank) allowing an easy insertion in V1 through the dura. Recording sites sample 640 μm in depth. B, Schematic of a Neuronexus Buzsáki64 (6 shanks) facilitating collection of data in the anteroposterior axis of dLGN. The recording channels span 180 μm. C, Example of a penetration in V1 on a sagittal section, Dye I is used to locate the electrode‘s track and is salient against backgrounds of DAPI staining. Using simultaneously the tissue slides (left) and the landmarks of the Allen Institute Atlas (Allen Institute for Brain Science, last panels on the right) allows to identify the lamination. D, E, Coronal sections of 2 mice, one with a penetration in dLGN (dLGN contours in dashed white) and another one in LP (LP contours in dashed yellow).

Figure 2.

Spike waveforms classification. A, Parameters used to classify waveforms: duration (between 2 peaks) and height ratio. B, C, Scatter of duration versus height ratios for all units in V1 aw (black) and dLGN aw (gray). The histogram of durations show multimodal distributions (Hartigan dip test, p < 0.001 for V1 and dLGN). Putative inhibitory neurons are seen for durations <0.3 in V1 and 0.2 in dLGN.

Figure 3.

State effect on firing rates and linearity in V1, dLGN, and LP. A, First column, Mean spontaneous and maximum evoked firing rates for the 3 areas and 2 states. Red represents the number of cells recorded for each condition. Box plots represent medians as a bar, mean as a cross, and illustrate 5th and 95th percentiles. Under anesthesia, spontaneous rate decreases only in dLGN (Mann–Whitney test, p = 0.0003). Maximum evoked firing rate is not showing statistical dependence on state in V1 and LP, but it is in dLGN (Mann-Whitney test, p = 0.03). Second and third columns, Firing rates by layer. Low spontaneous firing rate is seen in L2/3 in both states: Mann–Whitney test, L2/3 vs L4, p = 0.0043 (aw) and 0.0026 (an); L2/3 vs L5, p < 0.0001 (aw and an). Maximum firing rate levels do not vary with cortical layer and state: L2/3 vs L5: Kruskal–Wallis test, not significant (aw and an). B, F1/F0 index distributions in 3 areas. The number of units recorded are in red. Awareness does not alter spatial summation in V1 and LP. In dLGN, there is a significant shift toward more complex responses from anesthesia to awake state (solid gray; Mann–Whitney test, p < 0.0001). Few putative inhibitory cells (red) have a high value of F1/F0 ratio in V1 and dLGN. LP cells (light gray) are exclusively nonlinear. C, Distribution of F1/F0 index per cortical layers for all cells. In both states, L5 is the layer with the smallest mean of F1/F0 (Mann–Whitney test, aw: L2/3 vs L5: p = 0.0092; an: 0.0175). *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Figure 4.

Orientation and direction selectivity. A, Tuning curve examples from V1, dLGN, and LP, with high and low OSI (red). Blue lines indicate the mean spontaneous rate for each example cell in spikes/s. B, Mean OSI for 3 areas and 2 states. Cell count is in red. Box plots represent medians as a bar, mean as a cross, and illustrate 5th and 95th percentiles. A decrease in OSI is exclusively observed in anesthetized V1 (Mann–Whitney test, p = 0.0056). C, Laminar selectivity for orientation. L5 is the layer with the least orientation tuning, especially compared with layer 2/3 (Mann–Whitney test, aw: p = 0.0127; an: 0.0379). An increase in L4 cell selectivity is observed in awake mice (Red stars, Mann–Whitney test, p = 0.0072). D, Mean CV for excitatory and inhibitory neurons in V1 and dLGN. Putative inhibitory cells are less tuned for orientation than putative excitatory cells, in both areas (V1, Mann–Whitney test, p = 0.0029; dLGN, p = 0.0375). E, DSI does not significantly change with states (Kruskal–Wallis, not significant). Putative excitatory and inhibitory cells are grouped together when not explicitly separated into two groups. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Figure 5.

Spatial and temporal preferences. A, Representative examples of spatial and temporal frequency (SF and TF) tunings for the same cells seen in Figure 4A. B, Left, Box plots represent medians as a bar, mean as a cross, and illustrate 5th and 95th percentiles. Mean peak SF across layers is not significantly affected by state (Kruskal–Wallis test, not significant), (right) proportion of cells responding optimally to SF tested. C, Left, Mean peak TF is decreased in anesthetized dLGN (mean_: 6.27 ± 0.22 vs 4.07 ± 0.23 Hz; Mann–Whitney test, p < 0.0001). Right, proportion of cells responding optimally to TF tested. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Figure 6.

Three categories of contrast response curves. A, Representative examples of each category. Cells were classified HP when fitted with a sigmoid with or without plateau (2 top rows), bandpass (BP, third row) when fitted with a Gaussian or suppressed by contrast (SC, bottom row) when fitted with a negative sigmoid. B, Fraction of cells of each category in V1 and dLGN for both states. A higher percentage of SC cells are found in V1 aw vs V1 an (20% vs 11%).

Figure 7.

RF spatial characteristics. A, Examples of ON and OFF subfields maps at their peak response and after linear interpolation. Next, contours and overlap are depicted: blue represents OFF; red represents ON. B, RF size and ON-OFF radii. Throughout, box plots represent the 5th-95th percentile, bars represent medians, and crosses represent the means for each condition. RF size and radii are constant over states. LP RF size is on average 6 times bigger than those of dLGN. C, Subfield separation represents the distance between subfields centers. LP shows the biggest subfield separations between ON ad OFF subfields. OI (Overlap Index) is 1 if both ON and OFF overlap completely. The average Overlap Area and OI area are very similar for V1, dLGN, and LP (Kruskal–Wallis test, not significant).

Figure 8.

RF temporal characteristics. A, PSTHs are shown and illustrate the diversity of response time course (transient or sustained) of ON (red) and OFF (blue) responses corresponding to V1, dLGN, and LP cells seen in Figure 7A (in a different order). Bottom panel, Schematic representation of how the time to peak and sustained index were measured. B, Box plots represent medians as a bar, mean as a cross, and illustrate 5th and 95th percentiles. Mean of sustained index and time to peak values for each area and state. The sustained index is calculated as the ratio between the length of the response and that of the stimulus. Sustainedness is sensitive to state in dLGN (Mann–Whitney test, p = 0.005) and LP (Mann–Whitney test, p = 0.037). Time to peak is calculated from the PSTH as the time for the response to reach its maximum. Time to peak is extremely sensitive to anesthesia and is longer compared with responses in awake state for all areas. The number of subunits is in red. C, Top, Inhibitory cell sustained index is lower than excitatory neurons in the cortex (0.317 ± 0.018 vs 0.390 ± 0.01; Mann–Whitney test, p = 0.0009). Bottom, Cells with a sustained index >0.3 were classified as sustained, others as transient. The mean of time to peak for sustained (109.1 ± 2.9) and transient cells (90.2 ± 3.1, Mann–Whitney test, p < 0.0001). The number of subunits is in red. Putative excitatory and inhibitory cells are grouped together when not explicitly separated into two groups. *, p < 0.05; **, p < 0.01; ***, p < 0.001.