Animals sense the environment through pathways that link sensory organs to the brain. In the visual system, these feedforward pathways define the classical feedforward receptive field (ffRF), the area in space in which visual stimuli excite a neuron1. The visual system also uses visual context—the visual scene surrounding a stimulus—to predict the content of the stimulus2, and accordingly, neurons have been identified that are excited by stimuli outside their ffRF3,4,5,6,7,8. However, the mechanisms that generate excitation to stimuli outside the ffRF are unclear. Here we show that feedback projections onto excitatory neurons in the mouse primary visual cortex generate a second receptive field that is driven by stimuli outside the ffRF. The stimulation of this feedback receptive field (fbRF) elicits responses that are slower and are delayed in comparison with those resulting from the stimulation of the ffRF. These responses are preferentially reduced by anaesthesia and by silencing higher visual areas. Feedback inputs from higher visual areas have scattered receptive fields relative to their putative targets in the primary visual cortex, which enables the generation of the fbRF. Neurons with fbRFs are located in cortical layers that receive strong feedback projections and are absent in the main input layer, which is consistent with a laminar processing hierarchy. The observation that large, uniform stimuli—which cover both the fbRF and the ffRF—suppress these responses indicates that the fbRF and the ffRF are mutually antagonistic. Whereas somatostatin-expressing inhibitory neurons are driven by these large stimuli, inhibitory neurons that express parvalbumin and vasoactive intestinal peptide have mutually antagonistic fbRF and ffRF, similar to excitatory neurons. Feedback projections may therefore enable neurons to use context to estimate information that is missing from the ffRF and to report differences in stimulus features across visual space, regardless of whether excitation occurs inside or outside the ffRF. By complementing the ffRF, the fbRF that we identify here could contribute to predictive processing.
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We thank M. Mukundan, B. Wong and L. Bao for technical support; R. Beltramo for help with extracellular recordings; J. Isaacson, G. Keller, R. Nicoll and M. Heindorf for comments on the manuscript; the members of the Scanziani laboratory for discussions of this project as well as for comments on the manuscript; and M. Rio for software support. This project was supported by the National Institutes of Health grant U19NS107613, the Howard Hughes Medical Institute and the Swiss National Science Foundation grants P300PA_177882 and P2EZP3_162284 to A.J.K. and P300PA_177898 to M.M.R. Confocal images were acquired at the Nikon Imaging Center at the University of California San Francisco.
The authors declare no competing interests.
Peer review information Nature thanks Richard Born, Mark Hübener and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Extended data figures and tables
a, Experimental configuration. b, Top, schematics of stimuli used for size-tuning functions. Bottom, population-averaged size tuning of classical and inverse stimuli with sharp edges (left) and blurred edges (right) (Methods). Here and in all other figures, black and red traces are responses to classical and inverse stimuli, respectively, and shaded areas are periods of stimulus presentation. Solid lines are fits to the data (Methods). Triangles above size-tuning functions indicate the median preferred size for each condition. The inset show the maximum responses, with horizontal lines indicating the medians. Two-sided Wilcoxon signed-rank test were used to compare the maximum responses for classical and inverse stimuli under each condition; sharp edge, ***P = 2.0 × 10−9; blurred edge, ***P = 4.5 × 10−10; 773 neurons in 4 mice. Data are mean (traces or data points) ± s.e.m. (shading or error bars). Here and in all other figures, error bars are present but are sometimes smaller than symbols.
a, Classical(-only) neuron. Left, the response of a neuron probed with classical stimuli (black) increases with the size of the stimulus until it peaks at the preferred size of the neuron (top horizontal dotted line). The response then decreases owing to surround suppression (maximum suppressed level indicated by the lower dotted horizontal line). The response of the same neuron probed with inverse stimuli (red) starts at the maximally surround suppressed activity level (an inverse stimulus with a size of 0° is a full-field grating) and then decreases as the diameter of the grey patch increases, consistent with visual stimulation being progressively removed from the classical ffRF. Right, schematic of the ffRF of a neuron surrounded by its classical suppressive zone. b, Inverse-tuned neuron. Left, the response of the neuron probed with inverse stimuli (red) starts, as for the classical-only neuron, at the maximally surround suppressed activity level but then increases until it reaches the preferred inverse stimulus size of the neuron and decreases with larger diameters of the grey patch, consistent with visual stimulation being progressively removed from the fbRF. Right, schematic of the ffRF of a neuron surrounded by its fbRF. c, Four example stimuli: Two classical stimuli (1 and 2 of sizes x and y, respectively) and two inverse stimuli (3 and 4, also of sizes x and y, respectively). The inner dotted circle represents the outer border of the classical ffRF. The outer dotted circle represents the outer border of the suppressive region and, for inverse-tuned neurons, also the outer border of the fbRF. The response amplitudes to the four example stimuli (1 to 4) in a classical-only neuron and in an inverse-tuned neuron are marked in a and b, respectively, at the intersection between the green vertical lines (stimulus size) and the size-tuning functions.
a, Scatter plot of the peak responses of L2/3 excitatory neurons to classical and inverse stimuli (maximum responses to size-tuning curves in Fig. 1c). Classical and inverse median, 0.25 and 0.30 ∆F/F, respectively. Two-sided Wilcoxon signed-rank test; P = 7.7 × 10−5; same excitatory L2/3 neurons as in Fig. 1c; 1,190 neurons in 9 mice. b, Top, schematic of stimuli presented at different orientations to map the classical and inverse orientation preferences. We tested 8 orientations at intervals of 45° at the neuron’s preferred stimulus size and location using either a classical or an inverse stimulus. Bottom, calcium responses of two example neurons in V1 for different orientations using classical and inverse stimuli. c, Population-averaged tuning curve for inverse-tuned L2/3 excitatory neurons in response to classical and inverse stimuli. The preferred orientations of each neuron (independently for classical and inverse stimuli) were aligned to 0° and its activity was normalized to its maximum response (367 neurons in 4 mice). Solid lines are fits to the data (Methods). d, Tuning widths of orientation tuning curves obtained with classical stimuli compared with those obtained with inverse stimuli. For each neuron, tuning width was defined as the full width at half maximum (FWHM) of the fitted tuning curve. Two-sided Wilcoxon signed-rank test; P = 1.8 × 10−21; same neurons as in c. Green symbols represent the example neurons shown in b. e, Same as d but for orientation selectivity indices. The horizontal and vertical lines at 0.3 delimit the orientation-selective population. Two-sided Wilcoxon signed-rank test; P = 7.0 × 10−16; same neurons as in c. f, Same as d, e but for direction selectivity indices. Two-sided Wilcoxon signed-rank test; P = 0.46; same neurons as in c. g, Distribution of orientation offsets. For orientation-selective neurons only (see e, with both OSIs ≥ 0.3), an orientation offset was computed, defined as the absolute difference in orientation between the preferred orientation of a neuron for a classical and an inverse stimulus. h, Contrast response map. Classical and inverse stimuli were presented simultaneously, and different combinations of contrasts were tested. The contrast heat map was obtained by averaging normalized activity of inverse-tuned L2/3 excitatory neurons (86 neurons in 4 mice). Data are mean (traces or data points) ± s.e.m. (shading or error bars). i, Scatter plot of the peak responses of L4 excitatory neurons to classical and inverse stimuli (maximum responses to size-tuning curves in Fig. 1f). Classical and inverse median, 2.2 and 0.41 ∆F/F, respectively. Two-sided Wilcoxon signed-rank test; P = 2.5 × 10−7; same L4 neurons as in Fig. 1f; 35 neurons in 6 mice. j, Scatter plot of the peak responses of PV neurons to classical and inverse stimuli (maximum responses to size-tuning curves in Fig. 2a). Classical and inverse median, 0.40 and 0.48 ∆F/F, respectively. Two-sided Wilcoxon signed-rank test; P = 0.021; same PV neurons as in Fig. 2a, bottom; 60 neurons in 7 mice. k, Scatter plot of the peak responses of VIP neurons to classical and inverse stimuli (maximum responses to size-tuning curves in Fig. 2b). Classical and inverse median, 0.98 and 0.54 ∆F/F, respectively. Two-sided Wilcoxon signed-rank test; P = 3.6 × 10−4; same VIP neurons as in Fig. 2b, bottom; 74 neurons in 8 mice. l, Scatter plot of the peak responses of SOM neurons to classical and inverse stimuli (maximum responses to size-tuning curves in Fig. 2c). Classical and inverse median, 2.9 and 1.5 ∆F/F, respectively. Two-sided Wilcoxon signed-rank test; P = 1.3 × 10−23; same SOM neurons as in Fig. 2c, bottom; 179 neurons in 5 mice.
a, Top left, schematic of regular receptive field mapping. Stimulus diameter of 20° with a grid spacing of 15°. Centre left, trial-averaged calcium responses from an example neuron for each stimulus location. Bottom left, population-averaged receptive field for responses to classical or inverse stimuli aligned to the centre of the ffRF (489 neurons in 4 mice). Right, same but for fine receptive field mapping. Stimulus diameter of 10° with a grid spacing of 5° (only for part of the visual space covered with the regular mapping, see dotted rectangle on the left). b, Top, spatial offset of regular ffRF mapping compared to fine ffRF mapping (same 489 neurons in 4 mice). The ffRF centre of each neuron estimated by the fine grid mapping is aligned at [0,0] and the localization of its estimated ffRF centre estimated by the regular grid is plotted with respect to the fine grid estimated centre. Bottom, distribution of distances between the centre of ffRF estimated by fine grid mapping and the centre estimated by regular grid mapping (approximately 90% of neurons have a distance between the two centres of less than 10°). The green symbol represents the example neuron shown in a. c, Population-averaged receptive field for responses to classical or inverse stimuli aligned to the centre of the ffRF and only for L2/3 neurons that had a preferred ffRF size of more than 15° (319 neurons in 9 mice). d, Population-averaged size-tuning functions for classical (black: L2/3 neurons with ffRF >15°, 335 neurons in 9 mice; grey: L4 neurons, 35 neurons in 6 mice) and inverse (red: L2/3 neurons with ffRF >15°, 335 neurons in 9 mice; orange: L4 neurons, 35 neurons in 6 mice) stimuli. Solid lines are fits to the data (Methods). The triangles above size-tuning functions indicate the median preferred size for each condition. Data are mean (traces or data points) ± s.e.m. (shading or error bars).
a, Receptive field mapping of L5/6 units using classical and inverse stimuli. Top, experimental configuration. Electrophysiological recordings were obtained in awake mice. The silicon probe spanned all layers, including deep layers (see Methods for layer definition). Centre, receptive fields were mapped using classical and inverse stimuli. Bottom left, population-averaged ffRFs for L5/6 units. Bottom right, same for inverse stimuli, aligned relative to the centre of the ffRF (248 units in 20 mice). b, Population-averaged size tuning of L5/6 units using classical and inverse stimuli. Top, schematic of stimuli used for size-tuning functions. The classical and inverse stimuli were presented at the same location (within 10° of the estimated centre of the ffRF). Bottom, normalized size-tuning functions for classical and inverse stimuli. Solid lines are fits to the data (Methods). Triangles above size-tuning functions indicate the median preferred size for each condition. The inset shows maximum responses, with horizontal lines denoting the median values. Two-sided Wilcoxon signed-rank test; ***P = 1.1 × 10−4; 119 units in 20 mice. c, d, Same as a, b but for a subset of L5/6 units defined both as surround-suppressed and inverse-tuned (as compared with b, in which all L5/6 units that responded to at least one classical stimulus size were included (Methods)); 22 units in 12 mice (c); Two-sided Wilcoxon signed-rank test; *P = 0.016; 24 units in 12 mice (d). Data are mean ± s.e.m.
a, Experimental configuration for intrinsic imaging of V1 and HVAs. To estimate the visual area locations and their retinotopic maps using intrinsic imaging, we presented a narrow white bar (5°) on a black background, slowly drifting (10° per second) in one of the cardinal directions (‘Fourier’). We calculated the temporal phase of the Fourier component at the frequency of the bar presentation. This gave us the complete extent of V1. For locating HVAs, we cross-checked the Fourier maps with those obtained from the responses to 25° patches of gratings at different retinotopic locations (‘episodic’). b, Left, blood vessel pattern visible through the thinned skull. Centre, Fourier map of same field of view obtained with a vertical bar moving from nasal to temporal. Right, episodic map of the same field of view. c, Other example episodic maps. d, Experimental design to assess the effect of anaesthesia on V1 and HVAs. The responses to classical stimuli of neurons in an HVA, the LM or PM, and V1 were recorded using two-photon calcium imaging. The experiment started in awake mice by imaging either an HVA or V1. After induction of anaesthesia, the same neurons were imaged again. To reduce the influence of variability in anaesthesia levels, the first imaged area under anaesthesia was imaged again at the end of the experiment. e, Peak responses in visual areas. Top left, example calcium response of a neuron located in PM and another neuron located in V1 in an awake mouse (black) and responses of the same neurons in the anaesthetized mouse (grey). Top right, trial-averaged peak response for the same neurons shown on the left for an awake (black) and anaesthetized (grey) mouse. Bottom, same for a different mouse but recorded in V1 and the LM. f, Population-averaged peak responses in awake and anaesthetized mice. Top, population-averaged peak responses in V1, the LM and PM for awake (black) and anaesthetized (grey) mice. Two-sided Wilcoxon signed-rank test; V1, P = 6.2 × 10−40, 431 neurons in 5 mice; LM, P = 9.9 × 10−19, 106 neurons in 3 mice; PM, P = 1.1 × 10−10; 55 neurons in 2 mice. Bottom, population-averaged difference between normalized neuronal activity for the awake and the anaesthetized state. For each neuron, all responses were normalized by the peak activity in the awake state before computing the differences. Two-sided Wilcoxon rank-sum test; V1, 431 neurons in 5 mice; LM, 106 neurons in 3 mice; PM, 55 neurons in 2 mice. Comparison of V1 and LM (V1–LM), ***P = 1.2 × 10−25; V1–PM, ***P = 9.0 × 10−13; LM–PM, NS: P = 0.48. Data are mean (traces or data points) ± s.e.m. (shading or error bars).
a, Experimental configuration. A silicon probe was inserted in V1, spanning all cortical layers, in mice expressing channelrhodopsin 2 in inhibitory neurons (VGAT-ChR2). To assess the strength of inhibition of excitatory units when using the laser scanning technique (Fig. 5, Methods), the V1 recording site as well as seven other locations were scanned at 125 Hz. b, Raster plot of example excitatory unit in L5/6 in response to classical and inverse stimuli of 15° diameter under control conditions (30 trials each) and during silencing of V1 (blue; V1 sil.). Black and blue horizontal lines are periods of stimulus presentation and V1 silencing, respectively. Classical and inverse stimuli were presented in random order; trials with V1 silencing were randomized as well but are separated here for clarity. c, Reduction in firing of excitatory units. The reduction in firing was measured as 1 − the ratio between the optogenetic condition and the control condition. Silencing reached nearly 100% for both responses to classical and inverse stimuli, and for the baseline activity (26 units in 10 mice). d, Experimental configuration. To assess the effect of distance on the optogenetic stimulation of inhibitory units at the recording site, two medial and two lateral locations at 400 μm and 800 μm from the V1 recording site were targeted for laser stimulation while recording in V1. e, Modulation of the baseline of inhibitory units. The modulation index was defined as the difference between the activity during the optogenetic and the control condition divided by the sum of the two. The modulation index was high at the recording site (at 0 μm) and quickly dropped with distance (grey bars; two-sided Student’s t-test; 0 μm, ***P = 2.0 × 10−7; 400 μm, NS: P = 0.26; 800 μm, NS: P = 0.51; 16 units in 8 mice). As a comparison, the distance of the HVAs from the recording site is plotted on the same axis (black dots, right y-axis; 21 recording sites, 12 mice), suggesting that when pointing the laser at HVAs, direct activation of inhibitory neurons at the V1 recording site is unlikely. f, Experimental configurations. To assess the effect of the laser stimulation of HVAs on inhibitory units at the recording site, all 8 (top) or individual HVAs (bottom) were targeted for laser stimulation while recording in V1 (same configurations as during the experiments in Fig. 5 and Extended Data Figs. 8, 9). g, Modulation of the baseline of inhibitory units. The modulation indices were either negative or not significantly different from zero, indicating that the laser stimulation was unlikely to directly activate inhibitory neurons at the V1 recording site. Two-sided Student’s t-test; HVA, *P = 0.045; 16 units in 8 mice; M, NS, P = 0.16; 5 units in 4 mice; PM, NS, P = 0.24; 16 units in 8 mice; AM, NS: P = 0.11; 16 units in 8 mice; RL, NS: P = 0.46; 5 units in 4 mice; AL, NS: P = 0.051; 16 units in 8 mice; LM, NS: P = 0.064; 16 units in 8 mice; LI, *P = 0.015; 5 units in 4 mice; P, *P = 0.010; 5 units in 4 mice. h, Estimating the number of inhibitory neurons in HVA that project to V1. i, Methodology. A retrograde virus, AAVretro.CAG.Flex.tdTomato, was injected in V1 of GADcre mice to label glutamic acid decarboxylase (GAD)-expressing neurons projecting to the site of injection. j, Left, outlines of the cortical section where the confocal images shown on the right were acquired. The location of the imaged area is further indicated by the dotted square depicted on the outline. The rostro-caudal distance to bregma is indicated below the outline. Right, average intensity projection. Top right, DAPI staining highlights the higher density of neurons in L4 in V1 used to define V1 borders (white lines). Bottom right, the fluorescence of tdTomato reveals numerous cell bodies in V1 around the site of injection and even more distal in L1. k, Same as in j but only for the tdTomato fluorescence and for all HVAs targeted for laser stimulation in Fig. 5 and Extended Data Figs. 8, 9. White lines delimit the boundaries of the area. l, Quantification of tdTomato-positive neurons at the centre of the area. The number of tdTomato-positive neurons were counted in the section containing the centre of the investigated area. ‘HVAs’ represents the sum of tdTomato-positive neurons in all HVAs. Note the sparse inhibitory projections from HVAs to V1 but the abundance of local inhibitory projections within V1 (3 mice). Data are mean ± s.e.m.
a, Experimental configuration. A laser beam is scanned over HVAs around V1 for optogenetic silencing while recording in V1. b, Size-tuning function of an example unit (baseline subtracted firing rates) to classical stimuli with (blue) or without (black) HVA silencing. Note the relief of surround suppression at larger stimulus sizes upon silencing HVAs. c, Scatter plot of the classical suppression index with or without silencing of HVAs (Methods). Two-sided Wilcoxon signed-rank test; P = 0.033; 34 units in 12 mice. Closed and open symbols are units from L2/3 and L5/6, respectively. The green symbol represents the example unit shown in b. Data are mean ± s.e.m.
Extended Data Fig. 9 Silencing individual higher visual areas differentially affects responses to classical and inverse stimuli.
a, Schematic of results and experimental configuration. Individual HVAs are targeted for optogenetic silencing while recording in V1. b, Difference in firing rates (baseline-subtracted and normalized) between control conditions and individual HVA silencing for classical and inverse stimuli. Two-sided Wilcoxon signed-rank test; M, NS: P = 0.18; 22 units in 5 mice; PM, NS: P = 0.46; 42 units in 12 mice; AM, NS: P = 0.88; 42 units in 12 mice; RL, NS: P = 0.81; 22 units in 5 mice; AL, NS: P = 0.20; 42 units in 12 mice; LM, *P = 0.013; 42 units in 12 mice; LI, NS: P = 0.51; 22 units in 5 mice; P, *P = 0.020; 22 units in 5 mice. c, Scatter plot of the modulation indices of individual HVA silencing for responses to classical and inverse stimuli (Methods). Closed and open symbols are units from L2/3 and L5/6, respectively. Two-sided Wilcoxon signed-rank test; M, P = 0.033; 22 units in 5 mice; PM, P = 0.50; 42 units in 12 mice; AM, P = 0.47; 42 units in 12 mice; RL, P = 0.14; 22 units in 5 mice; AL, P = 0.19; 42 units in 12 mice; LM, P = 0.017; 42 units in 12 mice; LI, P = 0.067; 22 units in 5 mice; P, P = 0.039; 22 units in 5 mice. For the visual stimulus parameters used here, the LM showed the strongest effect in preferentially reducing responses to inverse stimuli. Data are mean ± s.e.m.
a, Left, experimental configuration. To localize V1 and the LM, we used intrinsic optical imaging (Methods). Right, response map to a nasal (magenta) and temporal patch of gratings (green). White lines represent area borders. b, Left, blood vessel pattern overlaid with area borders defined by the intrinsic map (black lines). The red-shifted calcium indicator RGECO1a was injected in V1 and GCaMP6f was injected in LM. Right, fluorescence of calcium indicators in V1 and the LM. The black square delimits the example imaging site shown in c. The scale is the same as in a. c, Left, the responses of LM boutons and of V1 cell bodies were recorded within the same cortical location. Centre, example imaging site of V1 cell bodies recorded 190 μm below the surface. The white square delimits the example imaging site shown on the right. Right, example imaging site of LM boutons in V1 recorded 110 μm below the surface. The white circles indicate the location of the example boutons in d and e. d, Top, schematic of receptive field mapping. Left, trial-averaged calcium responses from an example LM bouton aligned to its putative V1 target. Right, same but from an example bouton that is retinotopically offset with respect to its putative V1 target. e, Top, schematic of stimuli used for size-tuning functions. Left, right, trial-averaged calcium responses from the same example neurons as in d. f, Left, distance of population-averaged receptive field centre of V1 neurons from the centre of size-tuning stimuli (20 sites in 5 mice). Right, same for LM boutons. All average V1 receptive-field centres are located within 10° and average LM receptive field centres are more spread with larger standard deviations. g, Retinotopic spread measured as cumulative distance from population-averaged receptive-field centre. The ffRF centres of LM boutons (solid green line) were more retinotopically spread than V1 neurons measured over the same cortical surface (solid black line) or measured over approximately six times the surface of the LM bouton site (dotted black line). Two-sided Wilcoxon rank-sum test; LM–V1 same surface, ***P = 1.2 × 10−5; LM–V1 6 × surface, ***P = 3.1 × 10−4; LM, 311 boutons in 5 mice; V1 same surface, 530 neurons in 5 mice; V1 6 × surface, 2,352 neurons in 5 mice. h, Population-averaged size-tuning function of LM boutons (711 boutons in 5 mice) that are not retinotopically aligned with their V1 target. Both classical and inverse stimuli were presented at the ffRF location of their putative V1 targets (Methods) and not at the ffRF location of the imaged LM boutons. Solid lines are fits to the data (Methods). Triangles indicate the median preferred size. The insets display the maximum responses and horizontal lines denote the medians. Two-sided Wilcoxon signed-rank test; ***P = 1.4 × 10−11; 711 neurons in 5 mice. Data are mean ± s.e.m. i, Experimental configuration for two-photon calcium imaging in L2/3 neurons of the LM (green symbols) while presenting classical and inverse stimuli. j, Population-averaged size-tuning functions for classical and inverse stimuli. Solid lines are fits to the data (Methods). Triangles indicate the median preferred size. The insets display the maximum responses and horizontal lines denote the medians. Two-sided Wilcoxon signed-rank test; ***P = 4.7 × 10−10; 115 neurons in 3 mice. Data are mean ± s.e.m. k, Distribution of ITIs of LM (black) and V1 neurons (grey; same neurons as in Fig. 1c). Triangles above the distribution indicate medians. Two-sided Wilcoxon rank-sum test; ***P = 2.9 × 10−15; 115 neurons in 3 mice and 1,190 neurons in 9 mice for the LM and V1, respectively.
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Keller, A.J., Roth, M.M. & Scanziani, M. Feedback generates a second receptive field in neurons of the visual cortex. Nature (2020). https://doi.org/10.1038/s41586-020-2319-4