To encode specific sensory inputs, cortical neurons must generate selective responses for distinct stimulus features. In principle, a variety of factors can contribute to the response selectivity of a cortical neuron: the tuning and strength of excitatory1,2,3 and inhibitory synaptic inputs4,5,6, dendritic nonlinearities7,8,9 and spike threshold10,11. Here we use a combination of techniques including in vivo whole-cell recording, synaptic- and cellular-resolution in vivo two-photon calcium imaging, and GABA (γ-aminobutyric acid) neuron-selective optogenetic manipulation to dissect the factors that contribute to the direction-selective responses of layer 2/3 neurons in ferret visual cortex (V1). Two-photon calcium imaging of dendritic spines12,13 revealed that each neuron receives a mixture of excitatory synaptic inputs selective for the somatic preferred or null direction of motion. The relative number of preferred- and null-tuned excitatory inputs predicted a neuron’s somatic direction preference, but failed to account for the degree of direction selectivity. By contrast, in vivo whole-cell patch-clamp recordings revealed a notable degree of direction selectivity in subthreshold responses that was significantly correlated with spiking direction selectivity. Subthreshold direction selectivity was predicted by the magnitude and variance of the response to the null direction of motion, and several lines of evidence, including conductance measurements, demonstrate that differential tuning of excitation and inhibition suppresses responses to the null direction of motion. Consistent with this idea, optogenetic inactivation of GABAergic neurons in layer 2/3 reduced direction selectivity by enhancing responses to the null direction. Furthermore, by optogenetically mapping connections of inhibitory neurons in layer 2/3 in vivo, we find that layer 2/3 inhibitory neurons make long-range, intercolumnar projections to excitatory neurons that prefer the opposite direction of motion. We conclude that intracortical inhibition exerts a major influence on the degree of direction selectivity in layer 2/3 of ferret V1 by suppressing responses to the null direction of motion.
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We thank D. Whitney for help with analysis, A. Banerjee and F. Albeanu for help with patterned photostimulation setup, J. Chang for discussions about optogenetic stimulation, D. Ouimet for surgical assistance, N. Shultz and R. Satterfield for histology, T. Laviv for discussions about CyRFP, C. Baker and M. Bolton for cloning and the gift of the ChR2 construct, and the GENIE project for access to GCaMP6. This work was supported by EY011488, the Max Planck Florida Institute for Neuroscience, and the Max Planck Society.
Nature thanks A. Huberman and the other anonymous reviewer(s) for their contribution to the peer review of this work.
The authors declare no competing interests.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
Extended Data Fig. 1 Summed spine inputs fail to predict somatic direction selectivity, regardless of the method used to compute the sum.
a, No significant correlation between the DSI of summed spine inputs (with amplitude included) and somatic DSI. Spearman’s r = −0.11, P = 0.68, n = 17. b, No significant correlation between the fraction of spines that respond more strongly to the preferred direction and somatic DSI. Spearman’s r = −0.082, P = 0.75, n = 17.
Dashed line indicates cutoff of DSI > 0.3; n = 69 cells with spiking responses.
Figure shows responses to preferred, null and blank.
a, Difference in direction preference of excitation and inhibition are significantly greater than chance; Monte Carlo significance test, P = 0.023; difference in direction preference, 135° ± 95°, median ± IQR, n = 10 cells from 7 animals. b, FWHM of excitation and inhibition were not significantly different. FWHM 61° ± 46° and 61° ± 110° for excitation and inhibition, respectively; median ± IQR, n = 10, Wilcoxon sign-rank P = 0.70. c, Individual (grey) and population average (coloured) tuning curves for Ge, Gi and predicted Vm, peak-aligned to excitation.
Extended Data Fig. 5 I/E ratio at preferred direction is not correlated with simulated subthreshold direction selectivity.
Spearman’s r = 0.0061, P = 1, n = 10 cells from 7 animals.
Error bars, mean ± s.e.m.
a, Optogenetic suppression of GABAergic neurons significantly reduces spiking direction selectivity; Wilcoxon sign-rank, n = 14 cells with spiking responses, P = 0.0049. Black line, mean; grey lines, single cells. b, Absolute Vm depolarization induced by blue light is not related to optogenetic changes in Vm direction selectivity (computed as the difference in DSI between light off and light on conditions); Spearman’s r = 0.11, P = 0.70, n = 14 cells with spiking responses from 4 animals.
a, Underlying intrinsic signal polar direction map with direction-tuned GABAergic neurons overlaid. b, Direction preferences of inhibitory neurons and intrinsic signal direction preference map are significantly more similar than chance; P < 0.001, Monte Carlo significance test, n = 76 direction-selective neurons from 3 planes in 1 animal.
Extended Data Fig. 9 Reversal potential of optogenetically evoked PSPs is consistent with inhibition.
Grey points are individual data points; black is mean ± s.e.m. Data come from individual stimulation trials from one cell.
Grey points are individual data points; black is binned mean ± s.e.m. Data come from trial-averaged stimulation responses from n = 21 cells from 7 animals.
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Wilson, D.E., Scholl, B. & Fitzpatrick, D. Differential tuning of excitation and inhibition shapes direction selectivity in ferret visual cortex. Nature 560, 97–101 (2018). https://doi.org/10.1038/s41586-018-0354-1
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