Broad inhibition sharpens orientation selectivity by expanding input dynamic range in mouse simple cells

Abstract

Orientation selectivity (OS) is an emergent property in the primary visual cortex (V1). How OS arises from synaptic circuits remains unsolved. Here, in vivo whole-cell recordings in the mouse V1 revealed that simple cells received broadly tuned excitation and even more broadly tuned inhibition. Excitation and inhibition shared a similar orientation preference and temporally overlapped substantially. Neuron modeling and dynamic-clamp recording further revealed that excitatory inputs alone would result in membrane potential responses with significantly attenuated selectivity, due to a saturating input-output function of the membrane filtering. Inhibition ameliorated the attenuation of excitatory selectivity by expanding the input dynamic range and caused additional sharpening of output responses beyond unselectively suppressing responses at all orientations. This "blur-sharpening" effect allows selectivity conveyed by excitatory inputs to be better expressed, which may be a general mechanism underlying the generation of feature-selective responses in the face of strong excitatory inputs that are weakly biased.

Figure 1. Orientation selectivity of simple cells in layer 2/3 of the mouse V1

(A) Spike On and Off subfields of an example simple cell as examined by loose-patch recording. Top, arrays of post-stimulus spike-time histograms (PSTHs, generated from all the trials) for spike responses to unit On or Off stimuli. PSTHs were arranged according to the corresponding stimulus locations. Each pixel represents visual space of 5°. Red and green ovals depict the two-dimensional Gaussian fits of the On and Off subfield, respectively. Bottom, color maps for spike On (red) and Off (green) responses. The brightness of color represents the average evoked firing rate. The maps were smoothed by bilinear interpolation. The white line depicts the RF axis which passes through the centers of the On and Off subfields determined by the Gaussian fits. (B) Orientation tuning of the same cell in (A). Left, PSTHs for spike responses evoked by drifting sinusoidal gratings of various orientations. Arrow indicates the drifting direction of the grating. Right, orientation tuning curve as measured by the number of spikes evoked. The responses to gratings of opposite directions were averaged for each orientation. Red dash curve indicates the Gaussian fit, the peak of which indicates the preferred orientation angle θ. Error bar = SEM. (C) The relationship between θ and the orientation angle of RF axis. The red dash line is the identity line. The difference between θ +90° and the angle of RF axis is significantly smaller than 35° (p < 0.05, t-test, n = 34). (D) Subthreshold membrane potential (Vm, with spikes filtered out, left) and spike (AP, right) responses of another simple cell examined by whole-cell current-clamp recording. The red lines in the plots of Vm indicate the level of resting membrane potential. (E) The orientation tuning curves of Vm (top) and spike (bottom) responses for the cell in (D) and the corresponding Gaussian fits (red dash curves). Vm response was measured as the peak depolarization level relative to the resting membrane potential in the cycle-averaged waveform (first 3 cycles). Error bar = SEM. The average level of spike threshold of the cell is marked by the dotted line. (F) The plot of θ for spike responses versus that for Vm responses for the population of simple cells. The red dash line is the identity line. The difference between the two angles is significantly smaller than 15° (p < 0.001, t-test, n = 24). (G) The plot of OSI for spike responses versus that for Vm responses. The value for the cell in (E) is marked by the blue arrow. The red dash line is the identity line and the blue dash line is the best-fit linear regression line. R indicates the correlation coefficient. The black dash line labels OSI_AP = 1/3, which is a criterion for defining orientation-selective cells.

Figure 2. Orientation tunings of excitatory and inhibitory synaptic inputs to simple cells

(A) Vm responses to single drifting bars of various orientations. Red lines indicate the level of resting membrane potential. (B) One-dimensional RF of Vm responses to flashing bars of preferred orientation. Middle, average Vm responses evoked by bright (On) and dark (Off) bars at different spatial locations. Bar width was 3.5°. The solid and dash curves depict the spatial tuning curves (i.e. the envelope of peak response amplitudes) for On and Off responses, respectively. Bottom, superimposed On and Off spatial tuning curves. An arbitrary spike threshold (V_thr) of 22 mV above the resting level was applied. Inset, color maps for On (red) and Off (green) Vm responses before (left) and after (right) subtracting the spike threshold. The brightness of colors represents the peak amplitude of membrane depolarization. (C) Average excitatory (Ex) and inhibitory (In) currents evoked by bars of various orientations. (D) Orientation tuning curves for excitatory (red) and inhibitory (blue) conductances in the cell, fitted with a Gaussian function (dash curve). Error bar = SEM. (E) Plot of OSI of inhibitory input vs. that of excitatory input for 12 recorded simple cells. Solid symbol = mean ± SD. The red dash line is the identity line. OSI of inhibition is significantly lower than that of excitation (p < 0.001, paired t-test). (F) Average tuning curves (normalized) for excitatory input (Ex), inhibitory input (In) and the recorded Vm response (n=12). The tuning curves were aligned according to the preferred orientation angle, which is set as 0°. Error bar = SEM. Inset, average tuning width σ for excitation and inhibition. *, p < 0.01, paired t-test. (G) Plot of excitatory conductance vs. inhibitory conductance evoke by the same stimulus. The slope of the best-fit linear regression line is 1.9. Inset, distribution of I/E ratios. The I/E ratio was quantified as the ratio between the peak inhibitory and excitatory conductances for each orientation and then averaged for all orientations in each cell. Solid symbol = mean ± SD. (H) The preferred orientation angles of excitatory and inhibitory inputs relative to that of the Vm response, which is set as 0°. The values for the same cell are connected with lines. The inhibitory tuning curves in two cells were too flat so that the preferred orientation could not be determined. The difference between inhibition and excitation or between excitation and Vm is smaller than 20° (p<0.01, t-test).

Figure 3. Orientation tunings of output responses with and without inhibition

(A) Superimposed normalized excitatory (red) and inhibitory (blue) conductances evoked by bars of preferred and orthogonal orientations in the example cell shown in Figure 2 (marked by arrows). Inset, average conductance waveforms of all the recorded cells. Before averaging traces were aligned according to the onsets of excitatory responses. (B) Left, normalized orientation tuning curves of the same cell’s PSP responses derived from excitatory inputs alone (red), derived by integrating excitatory and inhibitory inputs (magenta), and experimentally recorded (black). Right, actual tuning curves of derived PSP responses. “a” and “b” with arrows mark the reduction of PSP response at preferred and orthogonal orientations, respectively. (C) OSIs of excitatory conductance (Ex), recorded Vm response (Vm), simulated Vm response with the excitatory input alone (Vm_simu(E)) and simulated Vm responses with integrating excitatory and inhibitory inputs (Vm_simu(E+I)). Solid symbol = mean ± SD. *, p < 0.01, ANOVA with post hoc test, n = 12. (D) Average normalized PSP tuning curves. Bar = SE, n = 12. (E) Reduction of PSP response at preferred and orthogonal orientations. Solid symbol = mean ± SD. *, p < 0.01, paired t-test. (F) OSIs of spike response (OSI_AP) for spikes recorded, spikes simulated by integrating excitatory and inhibitory inputs (Simu(E+I)), and those simulated with the excitatory input alone (Simu(E)). Solid symbol = mean ± SD. *, p < 0.001, ANOVA with post hoc test.

Figure 4. Membrane filtering and inhibitory sharpening of blurred selectivity

(A) Left, plot of peak amplitude of simulated PSP response vs. that of excitatory conductance (Ge) in two scenarios: without inhibition (black) and with co-variant inhibition (1X, 2X and 3X as strong as excitation). Ge₁ and Ge₂ are the amplitudes of two example excitatory inputs. ΔVm₁ and ΔVm₂, ΔVm₁’ and ΔVm₂’ are the amplitudes of corresponding PSP responses they generated when inhibition is excluded and included respectively. Inset, the first-order derivative of PSP-Ge function with (red, 2X) and without (black) inhibition. “p” (“parallel”) point marks where the two curves intercept. Right, temporal profiles of two model excitatory inputs (1nS and 2nS peak conductance) (top) and their corresponding PSP responses without inhibition (bottom). (B) The ratio of ΔVm₁/ΔVm₂ is consistently larger than that of Ge₁/Ge₂ (with 2X inhibition). Ge₁ and Ge₂ were sampled from 0.1nS to 10nS with an interval of 0.1nS, with Ge₂ always > Ge₁. The red dash line is the identity line. (C) The ratio of ΔVm₁/ΔVm₂ is consistently larger than that of ΔVm₁’/ΔVm₂’ (with 2X inhibition, in the range of 0.1–10nS). (D) Normalized tuning curves of simulated excitatory (red) and inhibitory (blue) inputs (left), of derived PSP responses without (Ge) and with (Ge+Gi) of inhibition (middle), and of derived spike responses (right). Excitation and inhibition evoked by the preferred stimulus is 1.5nS and 3nS respectively. Spike rates were derived according to a threshold-linear scheme. OSIs of the excitatory or inhibitory tunings were taken from the averaged values of our experimental results. (E) Schematic drawing of the design of dynamic clamp. The instantaneous Vm is sampled. The current injected into the cell (I_syn) is based on Vm and synaptic conductance. (F) The input-output curve for real cells in dynamic clamp recordings. PSP was measured as the peak amplitude of membrane depolarization in response to the injection of synaptic conductances, which were modeled the same as in (A). Inhibition was twice as strong as excitation. Data were presented as mean ± SD (n = 5 cells). Dashed lines mark injected excitatory conductances at 1nS and 2nS. Inset, example traces of recorded PSP responses in the absence (black) and presence (red) of inhibition (Ge = 2.5nS). (G) Normalized tuning curves of PSP responses (mean ± SD, n = 6 cells) in the absence (black) and presence (red) of inhibition in dynamic clamp recordings. The tunings of excitation (labeled by dashed curve, Ex) and inhibition were the same as in (D). Excitation and inhibition for the preferred stimulus were 2ns and 4nS respectively. Red data points except for the one at preferred stimulus are all significantly different from black data points (p <0.05, paired t-test).

Figure 5. Models of inhibitory mechanisms underlying OS

(A) Schematic drawing of the tuning curve of excitatory conductance, with conductance at orthogonal orientation about half of that at preferred orientation. (B) Left, the tunings of PSP response in the absence (black) and presence of inhibition (red). Circles and triangles label the PSP responses at preferred and orthogonal orientations respectively. “a” and “b” mark the suppression of the PSP response at preferred and orthogonal orientations respectively. Right, input-output function in the absence (black) and presence (red) of inhibition. Excitatory conductances at orthogonal and preferred orientations and their corresponding PSP responses are marked by dotted lines. Excitatory conductances fall into the initial phase of the input-output curve which could be approximated by a linear function f(x) = ax. (C) Similar plot as in (B) except that excitatory conductances fall into a more saturating phase of the input-output curve. (D) Left, the excitatory tuning (green curve) is fixed while varying the inhibitory tuning. Blue, untuned inhibition; red, broader inhibition; green, co-tuned inhibition, black, narrower inhibition. Middle, the corresponding PSP tuning curves. Dotted curve marks the PSP tuning in the absence of inhibition. Right, OSI of PSP responses vs. that of inhibitory inputs, with the excitatory tuning fixed.