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.

Although the functional properties of individual neurons in primary visual cortex have been studied intensely, little is known about how neuronal groups could encode changing visual stimuli using temporal activity patterns. To explore this, we used in vivo two-photon calcium imaging to record the activity of neuronal populations in primary visual cortex of awake mice in the presence and absence of visual stimulation. Multidimensional analysis of the network activity allowed us to identify neuronal ensembles defined as groups of cells firing in synchrony. These synchronous groups of neurons were themselves activated in sequential temporal patterns, which repeated at much higher proportions than chance and were triggered by specific visual stimuli such as natural visual scenes. Interestingly, sequential patterns were also present in recordings of spontaneous activity without any sensory stimulation and were accompanied by precise firing sequences at the single-cell level. Moreover, intrinsic dynamics could be used to predict the occurrence of future neuronal ensembles. Our data demonstrate that visual stimuli recruit similar sequential patterns to the ones observed spontaneously, consistent with the hypothesis that already existing Hebbian cell assemblies firing in predefined temporal sequences could be the microcircuit substrate that encodes visual percepts changing in time.

The neural code that relates the firing of neurons to the generation of behavior and mental states must be implemented by spatiotemporal patterns of activity across neuronal populations. These patterns engage selective groups of neurons, called neuronal ensembles, which are emergent building blocks of neural circuits. We review optical and computational methods, based on two-photon calcium imaging and two-photon optogenetics, to detect, characterize, and manipulate neuronal ensembles in three dimensions. We review data using these methods in the mammalian cortex that demonstrate the existence of neuronal ensembles in the spontaneous and evoked cortical activity in vitro and in vivo. Moreover, two-photon optogenetics enable the possibility of artificially imprinting neuronal ensembles into awake, behaving animals and of later recalling those ensembles selectively by stimulating individual cells. These methods could enable deciphering the neural code and also be used to understand the pathophysiology of and design novel therapies for neurological and mental diseases.

Accumulating evidence suggests that the olfactory bulbs (OBs) function as an independent circadian system regulating daily rhythms in olfactoryperformance.However,thecellsandsignalsintheolfactorysystemthatgenerateandcoordinatethesecircadianrhythmsareunknown. Using real-time imaging of gene expression, we found that the isolated olfactory epithelium and OB, but not the piriform cortex, express similar, sustained circadian rhythms in PERIOD2 (PER2). In vivo, PER2 expression in the OB of mice is circadian, approximately doubling with a peak around subjective dusk. Furthermore, mice exhibit circadian rhythms in odor detection performance with a peak at approximately subjective dusk. We also found that circadian rhythms in gene expression and odor detection performance require vasoactive intestinal polypeptide (VIP) or its receptor VPAC2R. VIP is expressed, in a circadian manner, in interneurons in the external plexiform and periglomerular layers, whereas VPAC2R is expressed in mitral and external tufted cells in the OB. Together, these results indicate that VIP signaling modulates the output from the OB to maintain circadian rhythms in the mammalian olfactory system.

Mounting evidence supports the hypothesis that the cortex operates near a critical state, defined as the transition point between order (large-scale activity) and disorder (small-scale activity). This criticality is manifested by power law distribution of the size and duration of spontaneous cascades of activity, which are referred as neuronal avalanches. The existence of such neuronal avalanches has been confirmed by several studies both in vitro and in vivo, among different species and across multiple spatial scales. However, despite the prevalence of scale free activity, still very little is known concerning whether and how the scale-free nature of cortical activity is altered during external stimulation. To address this question, we performed in vivo two-photon population calcium imaging of layer 2/3 neurons in primary visual cortex of behaving mice during visual stimulation and conducted statistical analyses on the inferred spike trains. Our investigation for each mouse and condition revealed power law distributed neuronal avalanches, and irregular spiking individual neurons. Importantly, both the avalanche and the spike train properties remained largely unchanged for different stimuli, while the cross-correlation structure varied with stimuli. Our results establish that microcircuits in the visual cortex operate near the critical regime, while rearranging functional connectivity in response to varying sensory inputs.