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. 2021 Jun 18;10:e68617. doi: 10.7554/eLife.68617

Figure 1. BLA neurons are activated during stimulus-outcome learning.

(a). Procedure schematic. CS, conditional stimulus (white noise or tone); O, outcome (sucrose solution or food pellet). (b) Schematic of fiber photometry approach for imaging bulk calcium activity in BLA neurons. (c) Representative fluorescent image of GCaMP6f expression and fiber placement in the BLA. (d) Schematic representation of GCaMP6f expression and placement of optical fiber tips in BLA for all subjects. Brain slides from Paxinos and Watson, 1998. (e) Representative examples of GCaMP6f fluorescence changes (Z-scored ∆F/F) in response to CS presentation (blue box), reward delivery, and reward retrieval (first food-port entry following reward delivery) across days of training. Traces from the last 6 days of training were selected from one of each two-session bin. See Figure 1—figure supplement 2 for raw GCaMP and isosbestic signal fluctuations. (f-g) Trial-averaged GCaMP6f fluorescence changes (Z-scored ∆F/F) in response to CS onset (f; blue) or reward retrieval during the CS (g; orange) across days of training. Shading reflects between-subjects s.e.m. Data from the last six sessions were averaged across two-session bins (3/4, 5/6, and 7/8). (h) Elevation [(CS probe entry rate)/(CS probe entry rate + preCS entry rate)] in food-port entries during the CS probe period (after CS onset, before first reward delivery), averaged across trials and across the 2 CSs for each day of Pavlovian conditioning. Gray lines represent individual subjects. (i-j) Trial-averaged quantification of maximal (i; peak) and area under the GCaMP Z-scored ∆F/F curve (j; AUC) during the 3 s period following CS onset or reward retrieval compared to equivalent baseline periods immediately prior to each event. Thin light lines represent individual subjects. N = 11 (see Figure 1—figure supplement 3 for data from N = 8 subjects with longitudinal data from each session). *p<0.05, **p<0.01 relative to pre-event baseline. See Figure 1—source data 1.

Figure 1—source data 1. Source data for Figure 1 and Figure 1—figure supplement 1–5.

Figure 1.

Figure 1—figure supplement 1. Food-port entry rate during Pavlovian conditioning for BLA fiber photometry GCaMP6f imaging experiment.

Figure 1—figure supplement 1.

Food-port entry rate (entries/min) during the CS probe period (after CS onset, before first reward delivery), averaged across trials and across the 2 CSs for each day of Pavlovian conditioning. Rats increased food-port approach responses to the CS across training (CS x Training: F(7,70) = 15.31, p<0.0001; CS: F(1,10) = 48.30, p<0.0001; Training: F(7,70) = 10.42, p<0.0001). ***p<0.0001, relative to preCS.
Figure 1—figure supplement 2. Representative examples of raw GCaMP6f and isosbestic fluorescent changes in response to cue presentation and reward delivery and retrieval across days of training.

Figure 1—figure supplement 2.

Raw GCaMP6f (470 nm channel) fluorescence and corresponding fitted fluorescent trace from the isosbestic (415 nm) channel.
Figure 1—figure supplement 3. BLA neurons are activated during stimulus-outcome learning across each of the eight Pavlovian conditioning sessions.

Figure 1—figure supplement 3.

(a–b) Trial-averaged quantification of maximal (a; peak) and area under the GCaMP Z-scored ∆F/F curve (b; AUC) during the 3 s following CS onset or reward retrieval compared to equivalent baseline periods immediately prior to each event from the N = 8 subjects for which we were able to obtain reliable recordings from each of the 8 Pavlovian conditioning sessions. Thin light lines represent individual subjects. Both CS and reward retrieval caused a similar elevation in the peak calcium response (Event v. baseline F(0.3,1.9) = 28.14, p=0.03; Training, Event type (CS/US), and all other interactions between factors, lowest p=0.12) and area under the calcium curve (AUC; Event v. baseline F(0.2,1.2) = 40.57, p=0.04, Training, Event type (CS/US), and all other interactions between factors, lowest p=0.21) across training. Analysis of each event relative to its immediately preceding baseline period confirmed that BLA neurons were robustly activated by both the onset of the CS as reflected in the peak calcium response (CS: F(1,7) = 9.95, p=0.02; Training: F(3.0,21.3) = 1.58, p=0.22; CS x Training: F(1.5,10.7) = 0.43, p=0.61) and AUC (CS: F(1,7) = 9.01, p=0.02; Training: F(2.3,16.0) = 0.56, p=0.60; CS x Training: F(1.5,10.2) = 0.30, p=0.68), as well as at reward retrieval during the CS [(Peak, Reward: F(1,7) = 12.22, p=0.01; Training: F(3.5,24.1) = 1.18, p=0.34; Reward x Training: F(2.5,17.4) = 1.75, p=0.20) AUC, Reward: F(1,7) = 13.73, p=0.008; Training: F(2.4,17.1) = 1.19, p=0.34; Reward x Training: F(3.0,21.3) = 2.46, p=0.09].
Figure 1—figure supplement 4. BLA reward responses aligned to reward delivery during Pavlovian conditioning.

Figure 1—figure supplement 4.

We detected a robust BLA response to reward retrieval during CS presentation during Pavlovian conditioning. This response was also detected when the data were aligned to reward delivery, which was signaled by the subtle but audible click of the pellet dispenser or sound of the sucrose pump. After initial training, reward retrieval often immediately followed reward delivery. (a) Trial-averaged GCaMP6f fluorescence (Z-scored ∆F/F) in response to reward delivery during the CS across days of training. Shading reflects between-subjects s.e.m. Data from the last six training sessions were averaged across 2-session bins (3/4, 5/6, and 7/8). (b) Trial-averaged quantification of maximal (peak) GCaMP Z-scored ∆F/F during the 3 s period following reward delivery compared to the equivalent baseline period 3 s prior to reward delivery. Thin light lines represent individual subjects. (c) Trial-averaged quantification of area under the GCaMP Z-scored ∆F/F curve (AUC) during the 3 s period following reward delivery compared to the equivalent baseline period. Across training, reward delivery caused a robust elevation in the peak calcium response (Reward delivery: F(1,10) = 57.73, p<0.0001; Training: F(2.5, 24.8)=1.29, p=0.30; Reward delivery x Training: F(1.8, 18.1)=0.43, p=0.64) and area under the calcium curve (Reward delivery: F(1,10) = 36.44, p=0.0001; Training: F(2.0, 19.7)=0.51, p=0.60; Reward delivery x Training: F(1.8,17.7) = 0.39, p=0.66). N = 11. *p<0.05, **p<0.01, ***p<0.001 relative to pre-event baseline.
Figure 1—figure supplement 5. Food-port entries during the CS in the absence of reward do not trigger a BLA response.

Figure 1—figure supplement 5.

We detected a robust elevation in BLA calcium activity in response to reward retrieval during CS presentation during Pavlovian conditioning. To determine the extent to which the action of entering the food-delivery port influenced this response, we examined BLA calcium activity in response to food-port entries during the CS probe period (after CS onset, before first reward delivery). Trial-averaged GCaMP6f fluorescence (Z-scored ∆F/F) in response to food-port entries during the CS across days of training. Shading reflects between-subjects s.e.m. Data plotted on the same scale as Figure 1 and 1-4 to facilitate comparison. Data from the last six training sessions were averaged across two-session bins (3/4, 5/6, and 7/8). N = 11.