Figure 7. Second US peak response akin to rapid post-CS pause excitation.

(A) Raster plot (upper panel) and average spike trace (lower panel) of an example IpN cell showing a second US peak (arrow). (B) Significant (plain orange) and non-significant (light orange) trial-by-trial correlation lines for cells showing both a first and a second US peak (n = 83). Pie chart shows the proportion of significant cells, black dotted line shows fit from a linear mixed model, integrating significant cells. (C) IpNs with a second US peak (Pk2) show lower average CR amplitudes at US onset than IpNs without (-), in both the first dataset (left boxplot pair; p<0.0001), as well as in the second dataset (right boxplot pair; p=0.021). (D) Average traces with SEM of spike activity relative to baseline for IpNs recorded without conditioned behavior on days 1–4 (black), and those recorded without conditioned behavior after day 4 (yellow). (E) Maximum spike rate velocity of the second US peak was significantly higher in recordings without CR behavior on day five and over (post-) compared to earlier recordings without behavior (pre-; p=0.0072). (F) Average spike traces of paired trials, aligned by CS pause minima (brown), and of US-only trials from the corresponding IpN cell, aligned by US pause (yellow) minima, for 12 recordings from the second dataset. The traces were standardized by the activity −150 to −50 ms relative to the pause minima. (G) Averages of the CS pause (brown) and US pause (yellow) traces in F, with SEM. For reference, US pause-aligned traces from paired trials of IpNs from the first dataset, with US pauses, without CR behavior, and from day 1–4, are shown in black. (H) The difference between US-responses in US-only trials after training (yellow) and US-responses in paired trials early in training before CR behavior is acquired (gray) highlight the absence of the first US peak and the substantial presence of a second peak after the US pause, in well-trained animals. Additionally, the difference between the CS pause-aligned traces from paired trials (brown) and the US-pause aligned traces from US-only trials of the same IpN set (yellow) suggests the two profiles only start to diverge substantially approx. 50 ms after the pause response.

Figure 7—figure supplement 1. IpN responses in paired trials and US-only trials, after optimal conditioning.

(A) Average spike traces during paired trials for 12 IpN cells with CS pause and CS-US facilitation (light red; average in plain red), and for 13 IpN cells that did not show both responses (gray; average in black). (B) Average spike traces for the same IpN cells in A, but during US-only trials. (C) The IpNs with CS pause and CS-US facilitation (red) showed substantially stronger post-US pause peak amplitudes (137.3 ± 89.4 Hz) than the IpNs without (black; 55 ± 53.9 Hz, p=0.0133, MWU). The second US peak was also stronger than the first US peak within the red IpN group (137.3 ± 89.4 vs 20.3 ± 62.3 Hz; p=0.0014, MWU), but not within the black IpN group (34.8 ± 32.8 vs 55.1 ± 53.9 Hz, p=0.4728, MWU).

Figure 7—figure supplement 2. Rapid post-CS pause excitation relates to broader subsequent excitation and to CR behavior.

(A) Average trial-by-trial correlation matrix (right panel) showing positive spike-eyelid position correlations for 16 IpN neurons with a CS pause, where a hotspot of positive correlations corresponding to rapid post-CS pause excitation (straddled by dotted white lines) shows a degree of separation from the hotspot corresponding to broader subsequent excitation. Average eyelid responses (left panel) correspond to the y-axis in the matrix; average spike traces (bottom panel) correspond to its x-axis. (B) Same as in A, here showing spike-eyelid velocity correlations. (C) Same as in A, here showing the auto-correlations among spike activity, which suggest a positive relation between the rapid post-CS pause excitation and broader subsequent excitation.