Visual stimuli recruit intrinsically generated cortical ensembles

The cortical microcircuit is built with recurrent excitatory connections, and it has long been suggested that the purpose of this design is to enable intrinsically driven reverberating activity. To understand the dynamics of neocortical intrinsic activity better, we performed two-photon calcium imaging of populations of neurons from the primary visual cortex of awake mice during visual stimulation and spontaneous activity. In both conditions, cortical activity is dominated by coactive groups of neurons, forming ensembles whose activation cannot be explained by the independent firing properties of their contributing neurons, considered in isolation. Moreover, individual neurons flexibly join multiple ensembles, vastly expanding the encoding potential of the circuit. Intriguingly, the same coactive ensembles can repeat spontaneously and in response to visual stimuli, indicating that stimulus-evoked responses arise from activating these intrinsic building blocks. Although the spatial properties of stimulus-driven and spontaneous ensembles are similar, spontaneous ensembles are active at random intervals, whereas visually evoked ensembles are time-locked to stimuli. We conclude that neuronal ensembles, built by the coactivation of flexible groups of neurons, are emergent functional units of cortical activity and propose that visual stimuli recruit intrinsically generated ensembles to represent visual attributes.There is a growing consensus in neuroscience that ensembles of neurons working in concert, as opposed to single neurons, are the underpinnings of cognition and behavior (1–3). At the microcircuit level, the cortex is dominated by recurrent excitatory connections (4, 5). Such densely interconnected excitatory networks are ideal for generating reverberating activity (1, 6, 7) that could link neurons into functional neuronal ensembles. Moreover, most cortical neurons are part of highly distributed synaptic circuits, receiving inputs from and projecting outputs to, thousands of other neurons (8, 9). In fact, the basic excitatory neurons of the cortex, pyramidal cells, appear to be biophysically designed to perform large-scale integration of inputs (10). All of these structural features indicate the possibility that rather than relying on the firing of individual neurons, cortical circuits may generate responses built out of the coordinated activity of groups of neurons. These postulated emergent circuit states could represent the building blocks of mental and behavioral processes (1–3, 11).In the visual cortex, there has been continuing progress in understanding functional properties and receptive fields of single neurons using single-unit electrophysiology and optical imaging (12–14). These single-neuron studies have provided a solid foundation for neuroscience. However, the focus on single neurons may provide an incomplete picture of this highly distributed neural circuit (3, 15). In fact, in recent years, the network activity patterns of the primary visual cortex (V1) in vitro and in vivo have been shown to be highly structured in spatiotemporal properties (13, 16–18). For example, using voltage-sensitive imaging, one can measure large-scale cortical dynamics with high temporal resolution, albeit without single-cell resolution (19–22). At this bird’s-eye view, wave-like spontaneous spatiotemporal patterns of activity appear similar to those patterns measured during visual stimulation (19, 21, 22). These findings imply that groups of neurons are active together in the absence of any visual input and that the same groups of neurons are also active together in response to visual stimulation. However, to test this hypothesis, one must measure the circuit activity with single-cell resolution.With calcium imaging, multineuronal activity can be visualized with single-cell resolution (16, 23), so it has become possible to discern exactly which neurons are activated under spontaneous and visually evoked conditions, cell by cell. Indeed, calcium imaging of brain slices from mouse visual cortex has revealed that groups of neurons become coactive spontaneously (24, 25) and that the same groups of neurons can be triggered by stimulation of thalamic afferents (26, 27). However, the patterns of activity found in slices may differ from the patterns of activity in vivo. Therefore, to determine the relation between spontaneous and evoked cortical activity patterns properly, it is necessary to measure them in vivo.Using two-photon calcium imaging in vivo, we have now mapped the spontaneous reverberating activity patterns in the V1 from awake mice with single-cell resolution and analyzed their relation to the activity patterns evoked by visual stimulation. We find patterns of coactive neurons that we term “ensembles,” defined as “a group of items viewed as a whole rather than individually” (28). Although the mere existence of these coactive neurons does not prove their functional importance, we provide converging lines of evidence that these ensembles are, in fact, functional units of cortical activity. This work provides a step in the progression of defining neuronal ensembles, rather than receptive fields of individual cells, as a building block of cortical microcircuits and suggests that these intrinsic neuronal ensembles are recruited when the cortex performs some of its most basic functions.