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, 2003). Thus, a possible cellular mechanism underlying memory consolidation in cortical neurons may involve the repeated activation of those spines that are activated during sensory stimulation. We showed that subthreshold single spine calcium signals are strictly NMDAR dependent, during both spontaneous (Figures 2A and 2B) and sound-stimulation-evoked activity (Chen et?al., 2011). In view of the essential role of spine calcium signaling (Nevian and Sakmann, 2006?and?Sabatini et?al., 2001) and NMDAR activation (Lee et?al., 2009?and?Matsuzaki et?al., 2004) for experience-dependent Bumetanide synaptic plasticity in cortical neurons, these patterned spine calcium signals during spontaneous up states are well suited as reinforcement signals underlying the cellular transformations associated with sleep-related memory consolidation (Diekelmann and Born, 2010, Marshall et?al., 2006?and?Stickgold, 2005). All experimental procedures were performed in accordance with institutional animal welfare guidelines and were approved by the state Cobimetinib molecular weight government of Bavaria, Germany. C57BL/6 mice (30�C50 postnatal days) were prepared under isoflurane anesthesia for two-photon calcium imaging and whole-cell patch-clamp recordings as described previously (Chen et?al., 2011, Chen et?al., 2012?and?Jia et?al., 2010). The patch pipette was used for loading the fluorescent calcium indicator, Oregon Green BAPTA-1 Hexapotassium (OGB-1, 120?��M), into layer 2 cortical pyramidal neurons in the primary auditory cortex. Dendritic spines were imaged in?vivo with the LOTOS procedure using a custom-built acousto-optic-deflector (AOD)-based two-photon microscope as recently described (Chen find more et?al., 2011?and?Chen et?al., 2012). In brief, the LOTOS-based procedure allowed acquirement of images at a frame rate of 1,000 frames/s?1 with 50?ns pixel dwell time and low excitation intensity. Auditory responses were evoked by 100?ms sound stimuli (10?ms rise/fall time) via an ES1 (Tucker-David Technologies) free-field speaker. For data analysis, the original image data that were acquired at a sampling rate of 1,000 frames/s?1 were downsampled 10- to 12-fold (to 80�C100 images s?1). Calcium signals were expressed as relative fluorescence changes (��f/f) corresponding to the mean fluorescence from all pixels within specified regions of interest (ROIs). The traces were further smoothed with an exponentially averaging IIR filter (time constant: 80?ms) as described previously ( Chen et?al., 2011?and?Jia et?al., 2010). In some recordings, calcium signals were observed in both dendrites and spines during action potential firing, which was not due to signals from the dendritic shafts scattering into the spine regions. This is because our procedure allows for significant separation of these signals ( Figure?S5). Statistical analyses were performed using paired or unpaired Student��s t test as appropriate. A p value