Extended Data Fig. 8: Supplementary analysis for in vivo electrophysiology.
From: Neurotensin orchestrates valence assignment in the amygdala
a–c: (a) A representative confocal image of a recording site in the BLA. (b) A summary of recording sites in the BLA from all mice. (c) A raster plot and histogram of a representative BLA neuron in response to sucrose-predictive cues. d: Both non-tagged BLA neurons and the BLA-CeA neurons showed significantly lower basal firing rates (in Hz) in the CRISPR group compared to the control group (Two-tailed unpaired t-test, non-projectors: **P = 0.0018; BLA-NAc: P = 0.6436; BLA-CeA: *P = 0.021). e: The CRISPR group showed significantly reduced port entry probability in sucrose trials, while enhanced freezing to CS during shock trials (Two-tailed unpaired t-test, sucrose trials: raw probability baseline, *P = 0.012; raw probability 10-25s post sucrose, ***P = 0.0006, shock trials: raw probability, P = 0.8894, z-score, *P = 0.0178). f, g: CRISPR mice showed overall reduced proportions, but not responsive amplitudes of excited and inhibited neurons to all three cues relative to the control group. Numbers in f indicate the total number of neurons that were excited and inhibited included in g. h: Functional clustering plotted separately for neurons in the control and CRISPR groups. i: Differences in proportions in neurons clustered in each functional cluster between the control and CRISPR groups. The proportion of BLA-NAc neurons in cluster 1 and 3 which preferentially encode positive valence was less in the CRISPR group compared to the control group. The proportion of BLA-CeA neurons in clusters 2 and 4 which preferentially encode negative valence was less in the CRISPR group compared to the control group. j: The positive valence encoding pattern observed in BLA-NAc neurons was abolished in the CRISPR group. The negative valence encoding pattern observed in BLA-CeA neurons was abolished in the CRISPR group (Two-tailed unpaired t-test, BLA-NAc: *P = 0.0483; BLA-CeA: *P = 0.0199). k–s: Unsupervised clustering of behavioral states. (k) First-order features were extracted from pose coordinates obtained from the videos. For each of these features, second-order features were extracted which were decomposed using PCA. Following PCA, t-distributed stochastic neighbor embedding (t-SNE) was performed on the top 30 principal components. A probability density function was computed over the 2-D t-SNE output and finally a watershed algorithm was used to form clusters. Each point in the t-SNE output plot represents a single trial. (l, p) Images depict the probability density function (PDF) estimate computed separately on t-SNE output for sucrose and shock trials, respectively. (m, q) The average latency to port in each sucrose trial cluster (m; One-way ANOVA, F6 = 20.924, ***P < 0.0001), and the average time spent freezing to CS (q, left) and average darting time (q, right) in each shock trial cluster (One-way ANOVA, freezing: F4 = 212.917, ***P < 0.0001, darting: F4 = 150.806, ***P < 0.0001). (n, r) Kernel density of changes of difference in distance to port in sucrose trials (n), and kernel density of time spent freezing (r, left panel) and time spent darting (r, right panel) in shock trials. (o, s) Control and CRISPR group showed vastly different neural responses to both sucrose and shock CSs in passive and active trials. In responses to sucrose CS, non-phototagged BLA neurons in the CRISRP group showed significantly smaller response in active trials, while the BLA-NAc neurons showed bigger response in passive trials and smaller response in active trials compared to the control group. In response to shock CS, non-phototagged BLA neurons in the CRISRP group also showed significantly smaller response in active trials, while the BLA-NAc neurons showed smaller response in active trials and the BLA-CeA neurons showed smaller response during passive trials compared to the control group (Two-way ANOVA with Holm-Sidak's multiple comparisons test. Sucrose CS: non-phototagged: P = 0.4523 for passive, *P = 0.0462 for active, BLA-NAc: *P = 0.0334 for both passive and active, BLA-CeA: P = 0.2969 for passive, P = 0.5054 for active. Shock CS: non-phototagged: P = 0.1716 for passive, *P = 0.0495 for active, BLA-NAc: P = 0.1145 for passive, *P = 0.0354 for active, BLA-CeA: *P = 0.0458 for passive, P = 0.8467 for active). See Methods for detailed statistical values. n and N denote number of neurons and mice in each group, respectively. Error bars and shaded regions around the mean indicate s.e.m.
