Eye-opening represents a turning point in the function of the visual cortex. Before eye-opening, the visual cortex is largely devoid of sensory inputs and neuronal activities are generated intrinsically. After eye-opening, the cortex starts to integrate visual information. Here we used in vivo two-photon calcium imaging to explore the developmental changes of the mouse visual cortex by analyzing the ongoing spontaneous activity. We found that before eye-opening, the activity of layer 2/3 neurons consists predominantly of slow wave oscillations. These waves were first detected at postnatal day 8 (P8). Their initial very low frequency (0.01 Hz) gradually increased during development to Ϸ0.5 Hz in adults. Before eye-opening, a large fraction of neurons (>75%) was active during each wave. One day after eye-opening, this dense mode of recruitment changed to a sparse mode with only 36% of active neurons per wave. This was followed by a progressive decrease during the following weeks, reaching 12% of active neurons per wave in adults. The possible role of visual experience for this process of sparsification was investigated by analyzing darkreared mice. We found that sparsification also occurred in these mice, but that the switch from a dense to a sparse activity pattern was delayed by 3-4 days as compared with normally-reared mice. These results reveal a modulatory contribution of visual experience during the first days after eye-opening, but an overall dominating role of intrinsic factors. We propose that the transformation in network activity from dense to sparse is a prerequisite for the changed cortical function at eye-opening.calcium waves ͉ cortical development ͉ mouse ͉ two-photon imaging ͉ up-down states

One of the challenges for modern neuroscience is to understand the rules of concerted neuronal function in vivo. This question can be addressed using noninvasive high-resolution imaging techniques like two-photon microscopy. This protocol describes a versatile approach for in vivo two-photon calcium imaging of neural networks, stained with membrane-permeant fluorescent-indicator dyes. It is based on a targeted pressure ejection of the dye into the tissue of interest and can be used for a large spectrum of indicator dyes, including Oregon Green 488 BAPTA-1 acetoxymethyl ester and Fura-2 acetoxymethyl ester. Through the use of dye mixtures and multicolor imaging, this technique allows the visualization of distinct neurons and glial cells up to 500 microm below the brain surface. It is suitable for staining the brain tissue of various different species (e.g., mouse, rat, cat and zebrafish) at all developmental stages. When combined with brain microendoscopy, it allows the monitoring of intracellular calcium signals in awake, behaving animals. The total time required to carry out the protocol, including dissection and cell staining, is approximately 2 h. Thereafter, imaging experiments might be performed for at least 6 h.

Fluorescent Ca(2+) indicator proteins (FCIPs) are attractive tools for studying Ca(2+) dynamics in live cells. Here we describe transgenic mouse lines expressing a troponin C (TnC)-based biosensor. The biosensor is widely expressed in neurons and has improved Ca(2+) sensitivity both in vitro and in vivo. This allows FCIP-based two-photon Ca(2+) imaging of distinct neurons and their dendrites in vivo, and opens a new avenue for structure-function analysis of intact neuronal circuits.

Non-technical summary The coherent activity of a large number of neurons forming functional cell assemblies crucially contributes to neuronal information processing in the brain. It is believed that activity of such populations constitutes a key mechanism linking awareness, stimuli from the external world, memory and behaviour. In this study, we recorded population calcium signals in the auditory cortex of mice using an optical fibre. We found that the population activity was dominated by transients lasting ∼1 s. These calcium transients directly reflected the action potential firing of local clusters of neurons and occurred spontaneously as well as in response to sound stimulation. We characterized in detail the spontaneous and sound-evoked calcium signals and the relation between the two. Thus, we were able to identify population calcium transients as an important element of sensory signalling.Abstract Population calcium signals generated by the action potential activity of local clusters of neurons have been recorded in the auditory cortex of mice using an optical fibre-based approach. These network calcium transients (NCaTs) occurred spontaneously as well as in response to sound stimulation. Two-photon calcium imaging experiments suggest that neurons and neuropil contribute about equally to the NCaT. Sound-evoked calcium signals had two components: an early, fast increase in calcium concentration, which corresponds to the short-latency spiking responses observed in electrophysiological experiments, and a late, slow calcium transient which lasted for at least 1 s. The slow calcium transients evoked by sound were essentially identical to spontaneous NCaTs. Their sizes were dependent on the spontaneous activity level at sound onset, suggesting that spontaneous and sensory-evoked NCaTs excluded each other. When using pure tones as stimulus, the early evoked calcium transients were more narrowly tuned than the slow NCaTs. The slow NCaTs were correlated with global 'up states' recorded with epidural potentials, and sound presented during an epidural 'down state' triggered a calcium transient that was associated with an epidural 'up state' . Essentially indistinguishable calcium transients were evoked by optogenetic activation of local clusters of layer 5 pyramidal neurons in the auditory cortex, indicating that these neurons play an important role in the generation of the calcium signal. Taken together, our results identify sound-evoked slow NCaTs as an integral component of neuronal signalling in the mouse auditory cortex, reflecting the prolonged neuronal activity of local clusters of neurons that can be activated even by brief stimuli.

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