Neuroscience

Valence and Salience Encoding in the Central Amygdala

Mi-Seon Kong
Ethan Ancell
Daniela M Witten
Larry S Zweifel author has email address

Department of Psychiatry and Behavioral Sciences, University of Washington, Seattle, USA
Department of Statistics, University of Washington, Seattle, USA
Department of Biostatistics, University of Washington, Seattle, USA
Department of Pharmacology, University of Washington, Seattle, USA

https://doi.org/10.7554/eLife.101980.1

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Figures and data

Acquisition of calcium signals during Pavlovian appetitive and fear conditioning.
A, CeA-GABAergic neurons were labelled with GCaMP6m, and their activity was recorded using a miniature microscope (Inscopix) via an implanted GRIN lens (left). A representative image of GCaMP6m expression and lens placement is shown on the right. Scale bar, 0.5 mm. B, Behavioral paradigm of the baseline, appetitive, and fear conditioning. To counterbalance the order of the two valence conditioning paradigms, there were two groups: appetitive → fear (5 mice) and fear → appetitive (5 mice) after the baseline session. C, Time spent near the food hopper during CS^Food (left, day: F = 7.422, P = 0.0005), latency to procure a pellet (middle, day: F = 20.41, P < 0.0001), and success rate (right, day: F = 14.69, P = 0. 0017) over 10 days of appetitive learning (mean ± s.e.m., grey lines represent individual data, n = 10). D, Freezing behavior during CS^Shock (trial: F = 13.49, P < 0.0001) throughout 10 trials of fear conditioning (mean ± s.e.m., grey lines represent individual data, n = 10). Detailed information about statistical results is provided in Supplementary Table 1.

Determination of CeA responses to appetitive and fearful stimuli.
A. Left: Example traces of CeA neurons that were responsive (upper three traces) and not responsive to food reward (bottom three traces). Right: Example traces of CeA neurons that were responsive (upper three traces) and not responsive to foot shock (bottom three traces). B, Step 1: Defining the “circular shift” operation. A neuron is randomly selected from the recorded neuron pool in our current study (denoted as Y). The calcium trace of the selected neuron is subjected to a circular shift operation, wherein it is moved from a randomly chosen time point (red bar). This operation results in a new calcium trace, as shown by the movement of the blue bar in Figure 2B; the traces that initially start with blue bars become red bar starting traces. C, Step 2: Creating a repository of test statistics for null data. A neuron is selected at random; then, the activity in each of its trials (20 trials for appetitive or 10 trials for fear conditioning, X = the number of trials) undergoes ‘circular shifting’, where a random point (red bar) is chosen within the circular shifting range. Then, on each trial, the Wilcoxon rank sum test between pre-stimulus vs. post-stimulus periods is computed. The final test statistic W_b^∗ representing a summary of all trials is obtained by summing the individual Wilcoxon rank sum test statistics over the trials with . Step 2 is repeated B times (we used B = 500), generating a null distribution of summed statistics . D, Step 3: calculating observed test statistics. Each observed neuron receives a summary test statistic obtained by summing the Wilcoxon rank sum test statistics from the individual trials. E, Step 4: Computing p-values. A p-value for the i^th neuron comes from a comparison of with . For the ith neuron, p_i⁺ (the number of divided by B+1) and p_i⁻ (the number of divided by B+1) are calculated. The final p-value for the i^th neuron (denoted as p_i) is 2 times the lesser of p_i⁺ and p_i⁻. A detailed description of this statistical analysis is provided in Methods.

Determination of CeA responses to appetitive and fearful stimuli.
A. Left: Example traces of CeA neurons that were responsive (upper three traces) and not responsive to food reward (bottom three traces). Right: Example traces of CeA neurons that were responsive (upper three traces) and not responsive to foot shock (bottom three traces). B, Step 1: Defining the “circular shift” operation. A neuron is randomly selected from the recorded neuron pool in our current study (denoted as Y). The calcium trace of the selected neuron is subjected to a circular shift operation, wherein it is moved from a randomly chosen time point (red bar). This operation results in a new calcium trace, as shown by the movement of the blue bar in Figure 2B; the traces that initially start with blue bars become red bar starting traces. C, Step 2: Creating a repository of test statistics for null data. A neuron is selected at random; then, the activity in each of its trials (20 trials for appetitive or 10 trials for fear conditioning, X = the number of trials) undergoes ‘circular shifting’, where a random point (red bar) is chosen within the circular shifting range. Then, on each trial, the Wilcoxon rank sum test between pre-stimulus vs. post-stimulus periods is computed. The final test statistic W_b^∗ representing a summary of all trials is obtained by summing the individual Wilcoxon rank sum test statistics over the trials with . Step 2 is repeated B times (we used B = 500), generating a null distribution of summed statistics . D, Step 3: calculating observed test statistics. Each observed neuron receives a summary test statistic obtained by summing the Wilcoxon rank sum test statistics from the individual trials. E, Step 4: Computing p-values. A p-value for the i^th neuron comes from a comparison of with . For the ith neuron, p_i⁺ (the number of divided by B+1) and p_i⁻ (the number of divided by B+1) are calculated. The final p-value for the i^th neuron (denoted as p_i) is 2 times the lesser of p_i⁺ and p_i⁻. A detailed description of this statistical analysis is provided in Methods.

Responses of CeA neurons during Pavlovian appetitive conditioning.
A, Top: Heat maps of CeA neurons aligned to CS^Food onset (top left, 1078 neurons) and head entry (top right) from day 1 of Pavlovian appetitive conditioning. Bottom: Heat maps of CeA neurons aligned to CS^Food onset (bottom left, 872 neurons) and head entry (bottom right) from day 10 of Pavlovian appetitive conditioning. Solid mint lines indicate CS^Food, and a dotted darker mint line represents food delivery. Black dotted line represents the first head entry after food delivery. B, Proportion of head entry responsive neurons between day 1 (top) and day 10 (bottom). C, Left: The average Z-scored activity of head entry-excited neurons on day 1 (147 neurons) and day 10 (307 neurons) of appetitive conditioning during -10 s to 10 s after the first head entry of each reward delivery. The dark lines and shaded areas indicate the mean and standard error of the mean (s.e.m.). Right: Average area under the curve (AUC) for the Z-scored activity after head entry on day 1 compared to day 10 of appetitive conditioning (0-5 s after head entry, mean ± s.e.m.). D, Left: The average Z-scored activity of head entry-inhibited neurons on day 1 (104 neurons) and day 10 (229 neurons) of appetitive conditioning during -10 s to 10 s after the first head entry of each reward delivery. Right: Average AUC for the Z-scored activity after head entry on day 1 compared to day 10 of appetitive conditioning (0-5 s after head entry). E, Left: Proportion of CS^Food -excited (orange), CS^Food -inhibited (blue), and not significant neurons (grey) on day 10 of appetitive learning (total 872 neurons). Right: Heat maps of CS^Food-excited neurons (top, n = 62) and CS^Food-inhibited neurons (bottom, n = 50) from all trials (20 trials). Solid mint lines indicate CS^Food onset and offset. F, The average Z-scored activity of each response type (orange, CS^Food-excited; blue, CS^Food-inhibited; grey, not significant). The dark lines and shaded areas represent the mean and s.e.m. The mint area represents 10 s of CS^Food. G, Left: Proportion of food-excited (orange), food-inhibited (blue), and not significant neurons (grey) on day 10 of appetitive learning (total 872 neurons). Right: Heat maps of food-excited neurons (top, n = 345) and food-inhibited neurons (bottom, n = 241) from all trials (20 trials). The dotted line indicates a food delivery H, The average Z-scored activity of each response type (orange, food-excited; blue, food-inhibited; grey, not significant). The dark lines and shaded areas represent the mean and s.e.m. ***P < 0.001, ****P < 0.0001. Detailed information about statistical results is provided in Supplementary Table 1.

Responses of CeA neurons during Pavlovian fear conditioning.
A, Heat maps of CeA neurons aligned to CS^Shock onset (519 neurons) from day 1 of Pavlovian fear conditioning. Solid orange lines indicate CS^Shock, and a dotted red line represents shock delivery. B, Left: Proportion of CS^Shock-excited (orange), CS^Shock-inhibited (blue), and not significant neurons (grey) on day 1 of fear conditioning (total 519 neurons). Right: Heat maps of CS^Shock-excited neurons (top, n = 19) and CS^Shock-inhibited neurons (bottom, n = 24) from all trials (10 trials). Solid orange lines indicate CS^Shock onset and offset. C, The average Z-scored activity of each response type (orange, CS^Shock-excited; blue, CS^Shock-inhibited; grey, not significant). The dark lines and shaded areas represent the mean and s.e.m. The orange area represents 10 s of CS^Shock. D, Left: Proportion of shock-excited (orange), shock-inhibited (blue), and not significant neurons (grey) on day 1 of fear conditioning (total 519 neurons). Right: Averaged heat maps of shock-excited neurons (top, n = 157) and shock-inhibited neurons (bottom, n = 62) from all trials (10 trials). A dotted red line represents shock delivery. E, The average Z-scored activity of each response type (orange, shock-excited; blue, shock-inhibited; grey, not significant). The dark lines and shaded areas represent the mean and s.e.m.

Salience and valence encoding in the CeA.
A, Among 872 neurons from appetitive conditioning and 519 neurons from fear conditioning, 303 neurons were registered during both learning. Heat maps of these 303 neurons during -10 s to 25 s after CS^Food onset from appetitive (left) and during -10 s to 25 s after CS^Shock onset from fear conditioning (right). All neurons are aligned to their activity to food. The same neuron is represented in the same row. Solid mint lines indicate CS^Food, and a dotted darker mint line represents food delivery, solid orange lines indicate CS^Shock, and a dotted red line represents shock delivery. B, Top: the proportion of significant neurons to food and shock (both; white), food-only (blue), shock-only (red), and not significant to both (grey). Bottom: detailed response types of all possible categories (excited, inhibited, not significant) for food vs. shock. White represents salience-encoding neurons showing the same response types to food and shock (n = 46, 15%), and black represents valence-encoding neurons showing different response types to food and shock (n = 198, 65%). Grey represents not significant neurons (n = 59, 20%). C, Salience-encoding neurons: the average Z-scored activity of neurons that were excited to both food and shock (n = 36, left) and inhibited to both food and shock (n = 10, right). Inserted bar graphs indicate average AUC of the Z-scored activity after food and shock delivery (0-10 s, mean ± s.e.m.). D, Valence-encoding neurons: the average Z-scored activity of neurons that were excited to food but inhibited to shock (n = 16, left) and inhibited to food but excited to shock (n = 25, right). Average AUC of the Z-scored activity after food and shock delivery are shown in inserted bar graphs (0-10 s, mean ± s.e.m.). E, The average Z-scored activity of neurons that were excited to food but not responsive to shock (n = 60, first), neurons that were inhibited to food but not responsive to shock (n = 50, second), neurons that were not responsive to food but excited to shock (n = 34, third), and neurons that were not responsive to food but inhibited to shock (n = 13, fourth). Inserted bar graphs indicate average AUC of the Z-scored activity after food and shock delivery (0-10 s, mean ± s.e.m.). The dark lines and shaded areas represent the mean and s.e.m. F, Proportion of significant neurons to CS^Food and CS^Shock (both; white), CS^Food-only (mint), CS^Shock-only (orange), and not significant to both (grey). G, Detailed response type of all possible categories (excited, inhibited, not significant) for CS^Food vs. CS^Shock. White represents salience-encoding neurons showing the same response types to CS^Food and CS^Shock (n = 7, 2%), and black represents valence-encoding neurons showing different response types to CS^Food and CS^Shock (n = 57, 19%). Grey represents not significant neurons (n = 239, 79%). *P < 0.05, ****P < 0.0001. Detailed information about statistical results is provided in Supplementary Table 1.

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