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Single neocortical neurons are driven by populations of excitatory inputs, which form the basis of neuronal selectivity to features of sensory input. Excitatory connections are thought to mature during development through activity-dependent Hebbian plasticity 1 , whereby similarity between presynaptic and postsynaptic activity selectively strengthens some synapses and weakens others 2 . Evidence in support of this process includes measurements of synaptic ultrastructure and in vitro and in vivo physiology and imaging studies 3-8 . These corroborating lines of evidence lead to the prediction that a small number of strong synaptic inputs drive neuronal selectivity, whereas weak synaptic inputs are less correlated with the somatic output and modulate activity overall 6,7 . Supporting evidence from cortical circuits, however, has been limited to measurements of neighbouring, connected cell pairs, raising the question of whether this prediction holds for a broad range of synapses converging onto cortical neurons. Here we measure the strengths of functionally characterized excitatory inputs contacting single pyramidal neurons in ferret primary visual cortex (V1) by combining in vivo two-photon synaptic imaging and post hoc electron microscopy. Using electron microscopy reconstruction of individual synapses as a metric of strength, we find no evidence that strong synapses have a predominant role in the selectivity of cortical neuron responses to visual stimuli. Instead, selectivity appears to arise from the total number of synapses activated by different stimuli. Moreover, spatial clustering of co-active inputs appears to be reserved for weaker synapses, enhancing the contribution of weak synapses to somatic responses. Our results challenge the role of Hebbian mechanisms in shaping neuronal selectivity in cortical circuits, and suggest that selectivity reflects the co-activation of large populations of presynaptic neurons with similar properties and a mixture of strengths.

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 excitatory 1-3 and inhibitory synaptic inputs 4-6 , dendritic nonlinearities 7-9 and spike threshold 10,11 . 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 spines 12,13 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.

In the mammalian cerebral cortex, neural responses are highly variable during spontaneous activity and sensory stimulation. To explain this variability, the cortex of alert animals has been proposed to be in an asynchronous high-conductance state in which irregular spiking arises from the convergence of large numbers of uncorrelated excitatory and inhibitory inputs onto individual neurons. Signatures of this state are that a neuron's membrane potential (Vm) hovers just below spike threshold, and its aggregate synaptic input is nearly Gaussian, arising from many uncorrelated inputs. Alternatively, irregular spiking could arise from infrequent correlated input events that elicit large fluctuations in Vm (refs 5, 6). To distinguish between these hypotheses, we developed a technique to perform whole-cell Vm measurements from the cortex of behaving monkeys, focusing on primary visual cortex (V1) of monkeys performing a visual fixation task. Here we show that, contrary to the predictions of an asynchronous state, mean Vm during fixation was far from threshold (14 mV) and spiking was triggered by occasional large spontaneous fluctuations. Distributions of Vm values were skewed beyond that expected for a range of Gaussian input, but were consistent with synaptic input arising from infrequent correlated events. Furthermore, spontaneous fluctuations in Vm were correlated with the surrounding network activity, as reflected in simultaneously recorded nearby local field potential. Visual stimulation, however, led to responses more consistent with an asynchronous state: mean Vm approached threshold, fluctuations became more Gaussian, and correlations between single neurons and the surrounding network were disrupted. These observations show that sensory drive can shift a common cortical circuitry from a synchronous to an asynchronous state.