Establishment of precise neuronal connectivity in the neocortex relies on activity-dependent circuit reorganization during postnatal development; however, the nature of cortical activity during this period remains largely unknown. Using two-photon calcium imaging of the barrel cortex in vivo during the first postnatal week, we reveal that layer 4 (L4) neurons within the same barrel fire synchronously in the absence of peripheral stimulation, creating a "patchwork" pattern of spontaneous activity corresponding to the barrel map. By generating transgenic mice expressing GCaMP6s in thalamocortical axons, we show that thalamocortical axons also demonstrate the spontaneous patchwork activity pattern. Patchwork activity is diminished by peripheral anesthesia but is mostly independent of self-generated whisker movements. The patchwork activity pattern largely disappeared during postnatal week 2, as even L4 neurons within the same barrel tended to fire asynchronously. This spontaneous L4 activity pattern has features suitable for thalamocortical (TC) circuit refinement in the neonatal barrel cortex.

Thalamocortical afferents innervate both excitatory and inhibitory cells, the latter in turn producing disynaptic feedforward inhibition, thus creating fast excitation-inhibition sequences in the cortical cells. Since this inhibition is disynaptic, the time lag of the excitation-inhibition sequence could be ∼2-3 ms, while it is often as short as only slightly above 1 ms; the mechanism and function of such fast IPSPs are not fully understood. Here we show that thalamic activation of inhibitory neurons precedes that of excitatory neurons, due to increased conduction velocity of thalamic axons innervating inhibitory cells. Developmentally, such latency differences were seen only after the end of the second postnatal week, prior to the completion of myelination of the thalamocortical afferent. Furthermore, destroying myelination failed to extinguish the latency difference. Instead, axons innervating inhibitory cells had consistently lower threshold, indicating they had larger diameter, which is likely to underlie the differential conduction velocity. Since faster activation of GABAergic neurons from the thalamus can not only curtail monosynaptic EPSPs but also make disynaptic ISPSs precede disynaptic EPSPs, such suppression theoretically enables a temporal separation of thalamically driven mono-and disynaptic EPSPs, resulting in spike sequences of 'L4 leading L2/3'. By recording L4 and L2/3 cells simultaneously, we found that suppression of IPSPs could lead to deterioration of spike sequences. Thus, from the end of the second postnatal week, by activating GABAergic neurons prior to excitatory neurons from the thalamus, fast feedforward disynaptic suppression on postsynaptic cells may play a role in establishing the spike sequences of 'L4 leading L2/3 cells'.

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In vivo calcium (Ca) imaging using two-photon microscopy allows activity to be monitored simultaneously from hundreds of individual neurons within a local population. While this allows us to gain important insights into how cortical neurons represent sensory information, factors such as photo-bleaching of the Ca indicator limit imaging duration (and thus the numbers of stimuli that can be tested), which in turn hampers the full characterization of neuronal response properties. Here, we demonstrate that using an encoding model combined with presentation of natural movies results in detailed characterization of receptive field (RF) properties despite the relatively short time for data collection. During presentation of natural movie clips to macaque monkeys, we recorded fluorescence signals from primary visual cortex (V1) neurons that had been loaded with a Ca indicator. For each recorded neuron, we constructed an encoding model that comprised an array of motion-energy filters that tiled over the RFs. We optimized the weight of each filter's output so that the linear sum of the outputs across the filters mimicked the neuron's Ca-signal responses. These models were able to predict the neural responses to a different set of natural movies with a significant degree of accuracy. Moreover, the orientation tunings of neurons simulated by the model were highly correlated with those experimentally obtained when grating stimuli were presented to the monkeys. The model predictions were also consistent with what is known about spatial frequency tunings, the structure of excitatory subfields of RFs (i.e., classical RFs), and functional maps for these RF properties in V1. Further analysis revealed a new aspect of V1 functional architecture; the extent and distribution of suppressive RF subfields varied among nearby neurons, while those for excitatory subfields were shared. Thus, applying our encoding-model analysis to two-photon Ca imaging of neuronal responses to natural movies provides a reliable and efficient means of analyzing a wide range of RF properties in multiple neurons imaged in a local region.