For each imaging field, neural responses were imaged to ten www.selleckchem.com/products/BKM-120.html whisker stimulations spaced 10 s apart. The analyses of changes in fluorescence were restricted to a 2 s window immediately following the onset of whisker stimulation. A total of 816 cells were imaged in seven fear-conditioned mice, and 833 cells in six explicitly unpaired control mice. Cortical networks are spontaneously active, and this spontaneous activity must be considered when defining evoked responses. To examine spontaneous activity we measured

changes in fluorescence in a 2 s time window immediately following each of ten sham whisker stimulations delivered with the same temporal pattern as during actual trials (Figure 3B and Movie S2). We used the resulting statistics of spontaneous activity for two purposes: (1) to examine if associative fear learning affected PF 2341066 spontaneous activity, and (2) to define thresholds of response magnitude (Figure 3C) and fidelity (Figure 3D) above which a neuron was considered responsive in subsequent trials with an actual stimulus. Here, mean response magnitude refers to the average fluorescent change across all ten sham stimuli, and fidelity refers to the number of sham trials out of ten that were temporally coincident with a given neuron’s spontaneous activity (see Experimental Procedures). Importantly, there were no significant differences in spontaneous

activity between paired and PI-1840 explicitly unpaired groups, as measured by mean response magnitude (Figure 3C: paired 1.17% ± 0.06%; unpaired 1.16% ± 0.03% dF/F, p = 0.14), mean response fidelity (Figure 3D paired 1.61; unpaired 1.66, p = 0.48) and network synchrony (Ch’ng and Reid, 2010 and Golshani et al., 2009) (Figure 3E, two-way ANOVA training effect F[1,320] = 1.4, p = 0.24). The values of spontaneous response magnitude (Figure 3C), and fidelity (Figure 3D) derived from sham stimuli were then used to determine the threshold for defining with 95% confidence whether a neuron was actually responding to

whisker stimulation or simply happened to be spontaneously active at the moment of whisker stimulation. For magnitude of response (dF/F), the 95% cutoff in paired mice was a 3.2% increase in fluorescence above baseline, and for explicitly unpaired mice was 2.7% above baseline (see gray shading in Figure 3C). For fidelity, the 95% cutoff was 4; that is, only 5% of cells were spontaneously active during the sham stimulus more than four out of ten trials (gray shading in Figure 3D). Using these thresholds, neurons could be confidently defined as responsive based on their mean response magnitude or based on the fidelity of their response. To determine whether associative learning impacts network coding of the CS we imaged cortical responses evoked by stimulation of the trained whisker (Figure 4 and Movie S3).