Spatial connectivity matches direction selectivity in visual cortex

Abstract

The selectivity of neuronal responses arises from the architecture of excitatory and inhibitory connections. In the primary visual cortex, the selectivity of a neuron in layer 2/3 for stimulus orientation and direction is thought to arise from intracortical inputs that are similarly selective^1-8. However, the excitatory inputs of a neuron can have diverse stimulus preferences^1-4,6,7,9, and inhibitory inputs can be promiscuous¹⁰ and unselective¹¹. Here we show that the excitatory and inhibitory intracortical connections to a layer 2/3 neuron accord with its selectivity by obeying precise spatial patterns. We used rabies tracing^1,12 to label and functionally image the excitatory and inhibitory inputs to individual pyramidal neurons of layer 2/3 of the mouse visual cortex. Presynaptic excitatory neurons spanned layers 2/3 and 4 and were distributed coaxial to the preferred orientation of the postsynaptic neuron, favouring the region opposite to its preferred direction. By contrast, presynaptic inhibitory neurons resided within layer 2/3 and favoured locations near the postsynaptic neuron and ahead of its preferred direction. The direction selectivity of a postsynaptic neuron was unrelated to the selectivity of presynaptic neurons, but correlated with the spatial displacement between excitatory and inhibitory presynaptic ensembles. Similar asymmetric connectivity establishes direction selectivity in the retina^13-17. This suggests that this circuit motif might be canonical in sensory processing.

Extended Data Figure 1. Targeted single neuron electroporation with survival control in vivo.

(a) Experimental pipeline: electroporation of the postsynaptic neuron, targeted by shadow-imaging and expression of GCaMP6; imaging of the postsynaptic neuron, labelled by dsRed; injection of the modified rabies virus; imaging and tracing of the presynaptic neurons marked by dsRed. (b-d) Schematic of the electroporation technique, performed under a two-photon microscope on a transgenic mouse expressing GCaMP6 in cortical excitatory neurons. (b)A pipette filled with DNA plasmids and Alexa 594 is targeted to a craniotomy; an 820 nm laser (red) excites Alexa 594 fluorescence (magenta) and GCaMP6 fluorescence (cyan). The latter is insensitive to neural activity because 820 nm is an isosbestic wavelength, where calcium-bound and calcium-free isoforms fluoresce approximately equally. (c) Upon electroporation, DNA plasmids and Alexa594 are transferred into a neuron expressing GCaMP6. (d). A healthy neuron maintains its GCaMP6 concentration and the resulting calcium-insensitive fluorescence (top), while a neuron with a damaged membrane bleeds indicator, gradually darkening and disappearing against the surrounding neuropil (bottom). (e) Time-lapse of an electroporation in layer 2/3 of mouse V1, using Alexa 594 negative contrast and calcium insensitive GCaMP6 fluorescence imaging: approach (left), electroporation (middle), pipette withdrawal (right). Scale bar 15 μm (f) The same neuron, imaged the next day at 920 nm, expressing the electroporated genes for dsRed (red) and maintaining healthy GCaMP6 expression (green). (g) Average fluorescence (mean ± s.e.m.) of somatic Alexa 594 (magenta) and GCaMP6 (cyan) relative to neuropil background (dashed line represents unity) before and after electroporation (arrow, n = 10 neurons). (h,i) Same as e,f, with a slower time-lapse of a neuron that survived the procedure. Images in h are 30 s long averages acquired 0-1 min (left), 2-3 min (centre) and 5-6 min (right) after electroporation. (j,k) Same as h,i, for a neuron that did not recover from the electroporation. (l) Same as g, for GCaMP6 somatic fluorescence (mean ± s.e.m.) in neurons that did (cyan, n=18) or did not (grey, n=10) survive the procedure. Scale bar 15 μm, same for all fluorescence images.

Extended Data Figure 2. Photoablation of supernumerary postsynaptic neurons before rabies injection.

(a) Cartoon of the protocol. The target neuron expresses both GCaMP6 (green) and dsRed (red), while surrounding neurons only express GCaMP6. The day after electroporation (Day1), the neuron is targeted with steady two-photon illumination at 820 nm, focused with intensity ~ 200 mW, for 10-20 s (top). The target neuron is ablated and by the next day (Day 2) it has disappeared (bottom). (b) Time lapse imaging during the photoablation of two neurons neighbouring neurons, lasting approximately 10 min. Imaging at 920 nm shows calcium sensitive fluorescence of GCaMP6 (top) and fluorescence of dsRed (bottom). Each of them was targeted with two photoablation pulses (red triangles). Photoablation pulses lasted 20 s, and each neuron was imaged for 30s afterwards. Each pulse increases cellular damage: localised photo-bleaching after the first pulse; elevated intracellular calcium and cell swelling after the second pulse. Neighbouring cells, not expressing dsRed, resist the photo-damage. (c) The successful elimination of the target neurons (red, see insets) is confirmed the day following the photoablation procedure: by Day 2 the target neuron has either disappeared (yellow arrow, top neuron) or gone in apoptosis (yellow arrow, bottom neuron). The surrounding tissue is unaffected, as shown by the normal activity detected in neighbouring neurons using Suite2p (blue-green ROIs and traces, scale bar 30 s and 10 s.d.). (d) Effectiveness of photoablation as a function of cortical distance from the photoablation target neuron (n = 9 attempts, mean ± s.e.).

Extended Data Figure 3. Time-course of rabies tracing and recordings.

(a) Viability of postsynaptic neurons as a function of day after the rabies virus injection, based on N = 17 injections. (b) Count of observed presynaptic neurons traced over the same period from N = 17 postsynaptic neurons (mean ± s.e.m.). (c) Fraction of viable presynaptic neurons (red) over the total traced (grey), and worst-case scenario population mortality of presynaptic neurons (dashed), estimated assuming the viability of each newly labelled presynaptic neuron degrades at the same rate as the viability of the postsynaptic neurons measured in a. (d) Average distribution across animals (N = 17, mean ± s.e.) of the imaging sessions used to record the responses of presynaptic neurons, split by cortical layer. The time of imaging did not systematically change across layer (red triangle, median, first and third quartiles), and most of the data was acquired before presynaptic neurons suffer from the toxicity of the rabies virus. (e) Distribution of responsivity of the presynaptic neurons across days (red violin plot with black median). Responsivity was measured as the maximum average stimulus-triggered response. To compare across sessions, presynaptic responsivity was normalised to the median responsivity of the local population (shaded line). (f) Longitudinal imaging of presynaptic neurons identified before (left, black ROI) and 5-12 days after (right, red ROI) rabies virus infection. Scale bar 25 μm. (g) Average responses to drifting gratings of the same presynaptic neurons before (black) and after (red) the rabies virus infection. Responses (4 s long) were z-scored with the respect to blank trials. Scale bar 1 std. (h) Preferred orientation of presynaptic neurons recorded before and after the rabies virus infection. At the time of recording used in this study, the preferred orientation of presynaptic neurons is unperturbed by the rabies virus infection (n = 51 neurons from N = 4 animals, circular correlation 0.76, p_r = 1.6*10^-6, Z-test).

Extended Data Figure 4. Classification of excitatory and inhibitory presynaptic neurons.

(a) Three example excitatory presynaptic neurons showing similar expression of dsRed (left) and decreasing expression of GCaMP6 (middle). The somatic mask obtained from dsRed and the GCaMP6 signal were used to compute a map of phase correlation in the 5 μm radius annulus around the somatic centre (right). In the first two examples, the stronger GCaMP expression in the soma compared to the surrounding neuropil results in positive peaks of the phase correlation. (b) Same as a for three example inhibitory neurons. The lack of GCaMP6 expression in the soma results in negative peaks of the phase correlation. (c) For each presynaptic neuron, the central peak of phase correlation is plotted against the standard deviation of the phase correlation within the 5 μm annulus around the soma (top). Darker dots indicate neurons whose identity was confirmed by immunostaining. Open dots represent example neurons shown in a-b. A classification boundary was used to split the clusters of excitatory (red) and inhibitory neurons (blue). This boundary was identified independently for each presynaptic ensemble with a bilinear fit. The average fit across datasets is shown (black line). The histogram (bottom) summarises the classification across experiments, with the average classification boundary. (d) Average expression of dsRed (left), GCaMP6 (middle) and map of phase correlation (right) for presynaptic neurons classified as excitatory neurons (top, n=516) or inhibitory neurons (bottom, n = 478). (e) Number of presynaptic neurons classified as inhibitory vs excitatory in experiments in CaMK2-GCaMP6 mice (red upward triangles, N=13) and GAD2-NLS-mCherry mice (red downward triangles, N=4). A linear fit (blue, r = 0.6, p_r=2.6*10^-3, linear correlation, F-test) shows that the fraction of traced presynaptic neurons that are inhibitory tends towards 30% as the yield of tracing increases; yet, due to the positive intercept, the fraction of inhibitory neurons may appear as high as 60% in experiments with low tracing yield. In control experiments where the red marker tdTomato was expressed only in excitatory neurons (AAV-CaMK2-tomato in CaMK2-GCaMP6 animals, grey circles), the fraction of neurons incorrectly classified as inhibitory was below 5%.

Extended Data Figure 5. Immuno-histochemical verification of presynaptic neuron classification method.

(a) Coronal maximum projection from a z-stack acquired in vivo, showing a section through a presynaptic network labelled with RV-dsRed (red) in a CaMK2a-GCaMP6s transgenic animal (green). Scale bar 50 μm. (b) Coronal brain slice matching the section in a. We could match n=94 out of the n=105 neurons traced in vivo. (c) Inset from b (dashed) showing the immunostaining for the inhibitory neurons marker GAD (blue). (d) ROC curve illustrating the sensitivity and specificity of the neuron classification method based on somatic GCaMP6 fluorescence (Extended Data Figure 4) against the ground-truth measurements obtained by immunostaining against GAD, for a range of classification boundaries. The fitted classification boundary was optimal (blue dot), yielding a specificity of 94% and sensitivity of 91%. (e) Two example presynaptic neurons classified as excitatory (n=49) from the z-stack in a. (f) Slice immunostaining of the two neurons in e. Neurons where confirmed excitatory if they expressed GCaMP6 and were GAD-negative (n = 45). Scale bar 40 μm. (g,h) Same as e,f, for two example presynaptic neurons classified as inhibitory. Neurons where confirmed inhibitory if they did not express GCaMP6 and were GAD-positive (n = 42).

Extended Data Figure 6. Spectral unmixing of dsRed and mCherry fluorescence.

(a) Emission spectra of GCaMP6, dsRed and mCherry. Shaded areas indicate the emission band captured by the green (G) and red (R) channels of the microscope. Note that the G channel collects mostly GCaMP6 fluorescence, while the R channel captures a mixture of mCherry, dsRed and GCaMP6 fluorescence. Emission spectra were normalised to their peak. (b) Two-photon action cross-section of dsRed (top, red) and mCherry (bottom, purple). Shaded bands indicate the excitation wavelengths used for imaging: 780, 890, 970, and 1020 nm. (c) Ratio between the two-photon action cross-sections of dsRed and mCherry (red) and its inverse (purple). The four wavelengths used for imaging (dark red) were chosen to maximise the SNR of each fluorophore while ensuring the ratio between the two signals was maximal. (d) Example field of view imaged at the four excitation wavelengths in the G channel. GCaMP6 was expressed in all neurons with an AAV2.1-Syn-GCaMP6s in a GAD-NLS-mCherry mouse; a subset of presynaptic neurons was traced with a dsRed rabies virus. (e) Same as d, for the R channel. (f) The fluorescence in the R channel (R _λ) plotted against the fluorescence in the G channel (G _λ) for each pixel, and for each excitation wavelength λ. Because the GCaMP6 labelling was dense and both the dsRed and the mCherry signals were sparse, and because the contribution of dsRed and mCherry to G_λ was minimal, the GCaMP6 signal contributing to R_λ could be recovered by piecewise robust linear regression (αG_λ + β). (g) The image representing the linear mix of dsRed or mCherry signals, F _λ, was recovered by subtracting the scaled GCaMP6 image from the R_λ. (h) An iterative algorithm was used to linearly unmix the two source images. Each unmixing iteration was constrained to minimise the quadratic reconstruction error over the data and return maximally uncorrelated sources R_dsRed and R_mCherry. (I,j) The two source images R_dsRed and R_mCherry for the example field of view in d-e. Note that the unmixing procedure correctly recovers the nuclear localisation of mCherry without any prior. Scale bars are 50 μm. Similar results were obtained for all GAD-NLS-mCherry mice (N=4).

Extended Data Figure 7. Distributions of individual excitatory and inhibitory presynaptic ensembles.

(a) Density of excitatory presynaptic neurons around the postsynaptic neuron (black triangle) as a function of cortical depth and horizontal distance from the postsynaptic neuron. Density was normalised to its maximum for display purposes. Depth marginals are shown on the left of each density map. (b) Same as a, for inhibitory presynaptic neurons. (c) Overlay of excitatory and inhibitory densities shown in a and b. Data are shown for the 12 experiments that included substantial recordings from L4.

Extended Data Figure 8. L2/3 neurons receive presynaptic excitation preferentially tuned to their orientation preference.

(a) Average tuning across the postsynaptic neurons responding to drifting gratings (N=16, mean ± s.e.m.), after alignment of their preferred stimulus direction to 0 deg. (b) Distribution of preferred direction relative to the postsynaptic preferred direction, for excitatory presynaptic ensembles across all layers (N=15, median ± m.a.d.). (c) Same as b, for excitatory presynaptic ensembles within L2/3 (top) and within L4 (bottom). (d) Same as b, for inhibitory presynaptic ensembles. (e) Average orientation tuning across the postsynaptic neurons responding to drifting gratings (N=16, mean ± s.e.m.), after alignment of their preferred stimulus orientation to 0 deg. (f) Average distribution of preferred orientation relative to the postsynaptic preferred orientation, for excitatory presynaptic neurons pooled across all layers (N=15, median ± m.a.d.). Presynaptic ensembles tuning for orientation (p_kw = 5*10^-8, two-sided one-way Kruskal-Wallis test) was significantly stronger than expected from random samples of the surrounding population (grey, median±m.a.d.). (g) Same as f, for presynaptic ensembles within L2/3 (top, N = 15, p_kw = 10^-7) and within L4 (bottom, N= 13, p_kw = 2*10^-5). (h) Same as f, for inhibitory presynaptic ensembles. (i) The tuning of the distributions of preferred orientation of excitatory presynaptic ensembles across layers plotted against the preferred orientation of their postsynaptic neuron. The co-tuning for orientation (r = 0.75, circular correlation, p_r = 4*10^-3, Z-test; p_v = 9*10^-8 circular V-test) was stronger than expected from random samples of the local population (p_{r_rand} < 10^-4 for circular correlation, p_{v_rand} < 10^-4 for V statistic). Upwards triangles represent experiments in CaMK2-GCaMP6 mice (N=11); downward triangles indicate experiments in GAD2-NLS-mCherry mice (N=4). (j) Same as i for excitatory presynaptic ensembles within L2/3 (top, r =0.92, p_r = 5*10^-3, p_v = 10^-5, p_{r_rand} = 2*10^-4, p_{v_rand} < 10^-4, N = 15) and within L4 (bottom, r = 0.76, p_r = 2*10^-2, p_v=3*10^-3, p_{r_rand} = 0.18, p_{v_rand} = 3*10^-3, N=13). (k) Same as i for inhibitory presynaptic ensembles. Inhibitory presynaptic ensembles were weakly biased to the orientation preference of the postsynaptic neuron (p_v = 0.05, circular V-test, N=4).

Extended Data Figure 9. Mapping retinotopy using individual neurons vs. widefield signals.

(a) The stimulus used for retinotopic mapping was a sparse random pattern of white and black squares on a grey background (top). The fluorescence time-course from the entire field of view was used to compute a global stimulus-triggered average response elicited by changes in luminance at each position. The centre of mass of this global receptive field (RF) was used to constrain the fits of widefield and neuronal RF to the appropriate retinotopic region. (b) Maximal projection from an example field of view. In this example, the field of view was subsampled in a grid of 9×9 regions of interest (ROIs, red squares) to compute widefield RFs. Scale bar: 100 μm. (c) The widefield RFs calculated for the ROIs in b, normalised to their maximum. The widefield RF centres from the grid of ROIs were interpolated to estimate a retinotopic map, assigning a widefield RF to each cortical location, whether it contained a responsive neuron, an unresponsive neuron, or neuropil. (d) Estimation of neuronal RFs. ON (red) and OFF (blue) receptive fields were estimated by regularised smooth pseudoinverse regression using either streams of white (ON) or black stimuli (OFF) as predictors, and assuming a common response kernel across neurons. ON and OFF subfields were then combined to estimate the RF centre (green dot). RF were considered significant if the cross validated correlation coefficient between predicted (red trace) and actual response (black trace) was greater 0.2. (e) Azimuth of neuron RF centre vs. widefield RF centre for all excitatory presynaptic neurons (black dots, n = 113, r_pre = 0.89, p _{r_pre} = 2.8*10^-39, linear correlation, F-test) and surrounding excitatory neurons (red density, n = 25677, r_all = 0.88 p _{r_all} < 10^-308, linear correlation F-test) across experiments. (f) Same as e for elevation (r_pre=0.80, p_{r_pre}= 9*10^-27; rall = 0.85, p _{r_all} < 10^-308). (g,h) Same as e and f for presynaptic inhibitory neurons (black dots, n = 37, r_pre=0.92, p_{r_pre}=1.3*10^-15, for azimuth; r_pre=0.71, p_{r_pre}=9.7*10^-7, for elevation) and all inhibitory neurons (n = 1963, r_all=0.95, p_{r_all} < 10^-308, for azimuth; r_all=0.74, p _{r_all} < 10^-308, for elevation).

Extended Data Figure 10. Spatial connectivity accords with direction selectivity in cortex and across layers.

(a) Distribution of excitatory and inhibitory presynaptic neurons in cortex, pooled across experiments (N=17), and polar tuning curves for each postsynaptic neuron (top). The colour hue indicates the average fraction of local excitatory (red) or inhibitory (blue) presynaptic neurons; the colour saturation indicates the max-normalised input density, averaged across experiments. (b,c) Same as a, plotting the excitatory and inhibitory presynaptic neurons separately. (d) Same as a, after rotating each presynaptic cortical distribution to align the postsynaptic preferred direction(N=16). The cortical angle of rotation, corresponding to the postsynaptic preferred direction, was calculated from the local retinotopic gradient at the postsynaptic location. After the alignment, the postsynaptic preferred orientation approximately maps to a line at the postsynaptic location (dashed line). (e) Same as d, for excitatory presynaptic neurons (f) Same as in d, for inhibitory presynaptic neurons. (g) Same as d, for the distribution of L1 and L2/3 excitatory and inhibitory presynaptic neurons in visual space, pooled across experiments after alignment to the preferred direction across postsynaptic neurons (N=16). (h,i) Same as g, distinguishing between excitatory and inhibitory presynaptic neurons. (j-l) Same as g-i, for presynaptic neurons in L4 and in superficial L5. In all panels, upwards triangles and circles represent CaMK2-GCaMP6 datasets; downward triangles and squares indicate GAD2-NLS-mCherry datasets.

Figure 1. Tracing the excitatory and inhibitory presynaptic inputs to an L2/3 pyramidal neuron.

(a) Time-lapse of electroporation (Day 0) and dsRed expression (Day 1) of the postsynaptic neuron. Scale bar: 20 μm. (b) Montage of Z-stack sagittal projections (taken 3 and 14 days after rabies injection) showing the postsynaptic neuron (yellow), its presynaptic ensemble (marked by dsRed, red) and the excitatory population (expressing GCaMP6, green). Lines indicate cortical layers. Scale bar: 100 μm. (c,d) Examples of an excitatory (Exc, n = 584) and an inhibitory (Inh, n = 426) presynaptic neuron in a CaMK2a-GCaMP6 mouse (N = 13): expression of dsRed (top) provides a somatic outline matching the expression of GCaMP6 (bottom) for the excitatory neuron but not for the inhibitory neuron. (e,f) Examples of an excitatory (Exc, n = 373) and inhibitory (Inh, n = 117) presynaptic neurons (top) in a GAD-NLS-mCherry mouse (N=4) injected with AAV-Syn-GCaMP6 (middle), where nuclear mCherry (bottom) distinguishes inhibitory from excitatory neurons. Scale bar 25 μm (g) Max-normalised density of presynaptic excitation pooled across experiments (N=17 postsynaptic neurons, 1,500 presynaptic neurons). All postsynaptic neurons resided in upper L2/3 (black triangles). (h) Same as g, for inhibition. (i) Overlay of the maps of excitation and inhibition. Hue indicates relative proportion of excitatory (red) vs inhibitory (blue) inputs, and saturation indicates max-normalised neuronal density. (j,k) Depth distributions for excitatory and inhibitory presynaptic neurons, for individual experiments (thin curves), and pooled data (thick curve). Vertical scale as in g-i. (l) Same as j,k, for the radial distributions of excitatory (red) and inhibitory (blue) presynaptic neurons, and their difference (black). Horizontal scale as in g-i.

Figure 2. Excitatory and inhibitory presynaptic ensembles are co-tuned for orientation but not direction.

(a) Peak-normalised responses to drifting gratings of an example postsynaptic neuron. Scale bar: 5 s. (b) Tuning curves of the postsynaptic neuron in a (neuron i) and of a second postsynaptic neuron (neuron ii), after alignment of their preferred direction to 0 deg (mean ± s.e., N = 10 trials). Preferred directions for the two neurons were-30 deg and 180 deg. (c) Average tuning across the postsynaptic neurons responding to drifting gratings (mean ± s.e., N=16). (d) Normalised responses of 12 example excitatory presynaptic neurons traced from the first postsynaptic neuron in a. (e) Distribution of preferred direction from the excitatory presynaptic ensembles connected to the postsynaptic neurons in a, relative to the postsynaptic preferred direction. (f) Average distribution of preferred direction for excitatory presynaptic neurons pooled across all layers (N=15, median ± m.a.d.). *** p_kw=10^-8, one-way Kruskal-Wallis test across orientations; n.s: p_w=0.39, Willcoxon signed rank test between preferred and opposite direction. (g) Same as e, for presynaptic ensembles within L2/3 (top) and within L4 (bottom). (h) Same as f, for presynaptic ensembles within L2/3 (top, N = 15, p_kw = 10^-7, p_w = 0.49) and within L4 (bottom, N=13, p_kw= 2*10^-5, p_w= 0.59). (i) Same as d, for 12 example inhibitory presynaptic neurons. (j) Same as e, for the inhibitory presynaptic ensembles (p_kw = 0.38, p_w = 0.25). (k) Same as f, for the average distribution of preferred direction of presynaptic inhibitory neurons (N = 4).

Figure 3. Elongated excitation and spatially offset inhibition accord with direction selectivity.

(a) Polar tuning curve of two postsynaptic neurons, showing preferred orientation (dashed) and direction (arrow). (b) Excitatory (red) and inhibitory (blue) presynaptic ensembles of postsynaptic neuron (open triangle, pointing in the preferred direction). The postsynaptic preferred orientation maps to a curve in cortex (dashed). Retinotopy is marked in 5 deg steps of azimuth (dark grey) and elevation (light grey). Coordinates: posterior (P), anterior (A), medial (M), lateral (L). (c) Excitatory presynaptic neurons in b replotted in visual space. Saturation indicates density. (d) Same as c, for inhibitory presynaptic neurons. (e) Average polar tuning curve across postsynaptic neurons, aligned to preferred direction (N= 16). (f) Excitatory and inhibitory presynaptic ensembles in visual space, pooled after alignment to the postsynaptic preferred direction (N=16). Hue indicates the relative proportion of excitation (red) vs. inhibition (blue); saturation indicates the average max-normalised density. (g) Same as f, for excitatory presynaptic neurons. (h) Same as f, for inhibitory presynaptic neurons. (i) Average postsynaptic tuning curve from e. (j) Angular density (shaded, mean ± s.e., N= 16) of excitatory (red) and inhibitory (blue) presynaptic neurons relative to the postsynaptic preferred direction. A sinusoid (capturing orientation selectivity) summed with a Gaussian (capturing direction selectivity) fit the data. (k) The angle of elongation of excitatory presynaptic ensemble correlates with the postsynaptic preferred orientation (r=0.72, p_r = 7*10^-3, circular correlation; p_v = 10^-4, circular V-test, N=16) significantly more than expected by chance (shaded area, median ± m.a.d., p_{r_rand} = 10^-3; p_{v_rand} < 10^-4). (l) Not so for the inhibitory presynaptic ensemble (r = -0.07, p_r = 0.77; p_v= 0.12). (m-o) Comparison of density in sectors opposite vs. ahead of the postsynaptic preferred direction for excitation (n, pw = 4*10^-2, N= 16, two-sided Wilcoxon signed-rank test), inhibition (o, p_w = 3*10^-3) and difference between excitation and inhibition (m, p_w = 4*10^-4). In b-o, upward triangles and circles indicate CaMK2-GCaMP6 datasets; downward triangles and squares indicate GAD2-NLS-mCherry datasets.

Figure 4. Spatially offset, delayed excitation and inhibition predict postsynaptic direction selectivity.

(a) Average excitatory and inhibitory presynaptic densities across experiments, showing contour at 10% of peak value. (b) In response to a visual stimulus, inhibitory currents (blue) rise in L2/3 pyramidal neurons later than excitatory currents (red). Traces indicate max-normalised changes in synaptic conductance measured in awake V1. Adapted from Ref. . (c) Simulated excitatory (red) and inhibitory (blue) presynaptic activity triggered by a bar sweeping in the postsynaptic preferred direction, as a function of time. The dotted line indicates the crossing of the bar with the position of the postsynaptic neuron (dashed line in a). Inhibitory activity is shown with the delay provided solely by spatial offset (dashed blue) and with the additional inhibitory lag (+τ, solid blue). (d) Net synaptic input (excitatory – inhibitory) to the postsynaptic neuron. (e-f) Same as c-d for a bar sweeping in the anti-preferred direction. (g) Same as a, for densities averaged across the three postsynaptic neurons with weakest direction selectivity (left) and for the three postsynaptic neurons with strongest direction selectivity (right). (h) The spatial offset between excitatory and inhibitory presynaptic ensembles (N=16) predicts the direction selectivity index measured from the postsynaptic neuron responses. Linear fit (red) with 95% confidence interval (grey), ** r =0.65, p_r =7*10^-3, F-test. Circles represent experiments from CaMK2-GCaMP6 mice; squares indicate experiments from GAD2-NLS-mCherry mice.