Reward conditioning increases inhibitory input onto Vglut1BLA→NAc neurons.

(A) Schematic of the cued sucrose conditioning task. A compound cue (tone and LED) predicts delivery of 10% sucrose to the center reward port 2 seconds after cue onset, followed by a 5 second consumption period, with an intertrial interval (ITI) of 10 seconds. (B) Average percentage of trials with a cued response (reward port entry within 7 seconds following cue presentation) across 5 training sessions (n = 6 mice). (C) Average latency to initiate a cued response across 5 training sessions from the same mice in B. (D) Average time-resolved z-scored probability of port entry aligned to cue onset across training sessions from the same mice in B. (E) Schematic of experimental design for fluorescently labeling BLA→NAcVglut1 neurons using a retrograde AAV expressing Cre-dependent eGFP in Vglut1-Cre mice. (F) Average spontaneous action potential firing frequency of identified Vglut1BLA→NAc neurons in naïve control mice and reward conditioned mice (n = 15 cells/group from 3 mice/group; p =0.0529, unpaired two-tailed t test). (G) Average spontaneous inhibitory post-synaptic current (sIPSC) amplitude in Vglut1BLA→NAc neurons in naïve and conditioned mice (n = 15 cells/group from 3 mice/group; ****=p < 0.0001, unpaired two-tailed t test). (H) Average sIPSC frequency in Vglut1BLA→NAc neurons in in naïve and conditioned mice (n = 15 cells/group from 3 mice/group; ****=p < 0.0001, unpaired two-tailed t test). (I) Average resting membrane potential (Vm) of Vglut1BLA→NAc neurons in naïve and conditioned mice (n = 20-24 cells/group from 3 mice/group ****=p < 0.0001, unpaired two-tailed t test). (J) Average first spike latency in response to current injection in Vglut1BLA→NAc neurons in naïve and conditioned mice (n = 20-24 cells/group from 3 mice/group; *=p < 0.05, two-way ANOVA w/ Holm-Sidak’s multiple comparisons test). (K) Average number of evoked action potentials in response to current injection in Vglut1BLA→NAc neurons in naïve and conditioned mice (n = 20-24 cells/group from 3 mice/group; *=p < 0.05, two-way ANOVA w/ Holm-Sidak’s multiple comparisons test).

Chemogenetic inhibition of Vglut1BLA→NAc neurons enhances cued sucrose responding and cue-evoked approach behavior.

(A) Schematic illustrating the viral strategy used to selectively target Vglut1-expressing BLA→NAc projection neurons for chemogenetic inhibition. Vglut1-Cre mice received retrograde AAVrg-DIO-Flp (or control virus, AAVrg-DIO-EGFP) in the nucleus accumbens (NAc) and a Flp-dependent AAV-fDIO-hSyn-hM4Di-mCherry in the basolateral amygdala (BLA), restricting hM4Di expression to Vglut1BLA→NAc projection neurons (Vglut1-hM4DiBLA→NAc). (B) Representative coronal brain section showing mCherry-labeled hM4Di expression in the BLA of Vglut1-Cre using the approach described in (A). (C) Representative whole-cell current-clamp recordings from hM4Di-expressing BLA neurons showing spontaneous firing before and after bath application of the DREADD agonist Compound 21 (C21). (D) Quantification of firing frequency before (Pre) and after (Post) C21 application in hM4Di-expressing BLA neurons (**p<0.01, paired one-tailed t test; n = 8 cells from 2 mice). (E) Schematic of the cued sucrose conditioning task. An auditory cue (2 seconds) predicted delivery of 10% sucrose (5 seconds), followed by an inter-trial interval (ITI; 10 seconds). (F) Percentage of trials with a cued response across five training sessions for control mice (black) and Vglut1-hM4DiBLA→NAc mice (pink), (**p<0.01, two-way ANOVA with Holm–Sidak’s multiple comparisons; n = 8–11 mice/group). (G–I) Average time-resolved Z-scored probability of port entry aligned to cue onset for control (black/gray) and Vglut1-hM4DiBLA→NAc (pink) mice on training day 1 (G), day 3 (H), and day 5 (I), (**p<0.01, two-way ANOVA with Holm–Sidak’s multiple comparisons). Shaded areas indicate mean ± SEM.

Chemogenetic inhibition of Vglut1BLA→NAc neurons enhances instrumental reward responding.

(A) Schematic illustrating the self-paced sucrose reward task. In the behavioral task, mice initiated trials via an initiation port, triggering a brief cue (2 seconds) followed by delivery of 10% sucrose (5 seconds) and an inter-trial interval (ITI, defined by next initiation port entry). (B) Mean number of initiation port entries across five training sessions for control mice (black) and Vglut1-hM4DiBLA→NAc mice (pink), (*p<0.05, two-way ANOVA with Holm– Sidak’s multiple comparisons; n = 8–11 mice/group). (C) Mean number of reward port entries across training sessions from the same mice shown in B, (*p<0.05, two-way ANOVA with Holm–Sidak’s multiple comparisons). (D–F) Average time-resolved Z-scored probability of reward port entry aligned to the initiation poke for control (black) and Vglut1-hM4DiBLA→NAc (pink) mice on training day 1 (D), day 3 (E), and day 5 (F). Shaded regions indicate mean ± SEM.

Chemogenetic inhibition of Vglut1BLA→NAc neurons enhances reward port entry without altering discrimination or reversal learning performance.

(A) Mean number of reward port entries for cherry-flavored 10% sucrose and grape-flavored 5% sucrose across training sessions in control and Vglut1-hM4DiBLA→NAc mice. The dotted line indicates the reversal session, after which the location of the solutions were switched. (B) Combined reward port entries (collapsed across flavors) across training sessions for control (black) and Vglut1-hM4DiBLA→NAc mice (pink), (*p<0.05, n=6-10 mice/group). (C) Cherry taste preference index across training sessions for control and Vglut1-hM4DiBLA→NAc mice, calculated as the normalized difference in reward port entries between cherry- and grape-flavored solutions. Data are shown as mean ± SEM.

Chemogenetic inhibition of Vglut1BLA→NAc neurons biases reward valuation toward a previously less-preferred option.

(A) Schematic of the behavioral timeline. On Day 1, mice were given free access to two flavored sucrose solutions (5% sucrose with grape flavor vs. 10% sucrose with cherry flavor) to assess baseline preference following saline injection. On Days 2 and 3, mice received the DREADD agonist C21 (0.1 mg/kg, i.p.) prior to exposure to only the 5% sucrose grape solution. On Day 4, mice again received saline and were given free access to both sucrose solutions to assess preference following conditioning. (B–C) Average cumulative reward port entries over time (B) and total reward port entries (C) on Day 1 in saline-treated control mice (n = 8 mice; ***p < 0.001, unpaired one-tailed t test). (D–E) Average cumulative reward port entries (D) and total reward port entries (E) on Day 1 in Vglut1-hM4DiBLA→NAc mice (n = 11 mice; ***p < 0.001, unpaired one-tailed t test). (F) Cherry preference index on Day 1 in control mice and Vglut1-hM4DiBLA→NAc mice (ns, unpaired one-tailed t test). (G–H) Average cumulative reward port entries (G) and total reward port entries (H) on Day 4 in control mice (n = 8 mice; ***p < 0.001, unpaired one-tailed t test). (I–J) Average cumulative reward port entries (I) and total reward port entries (J) on Day 4 Vglut1-hM4DiBLA→NAc mice (n = 11 mice; unpaired one-tailed t test). (K) Grape preference index on Day 4 in control mice and Vglut1-hM4DiBLA→NAc mice (*p < 0.05). Individual data points represent individual mice. Data are shown as mean ± SEM.

Chemogenetic inhibition of Vglut1BLA→NAc neurons enhances auditory fear discrimination.

(A) Schematic of the discriminative auditory fear conditioning and recall paradigm. Mice received the DREADD agonist Compound 21 (C21; 0.3 mg/kg, i.p.) 15 min prior to conditioning. During conditioning, mice were exposed to two auditory cues: a white noise (CS−, 60 dB) and a 5 kHz tone (CS+, 90 dB) paired with footshock. Twenty-four hours later, freezing behavior was assessed during presentation of CS− and CS+ in a distinct context. (B) Time-resolved freezing behavior during fear recall for control mice (black) and Vglut1-hM4DiBLA→NAc mice (pink). Shaded bars indicate cue presentation epochs (grey = CS-, pink = CS+). (C) Total freezing time during the recall session collapsed across cue presentations, showing reduced freezing in mice with chemogenetic inhibition of Vglut1BLA→NAc neurons relative to controls (*p < 0.05, unpaired two-tailed t-test). (D) Mean freezing time during baseline, CS−, and CS+ periods for control and Vglut1-hM4DiBLA→NAc mice, (*p < 0.05, 2-way ANOVA w/ Dunnett’s multiple comparisons test). (E) Discrimination index [(CS+ − CS−)/(CS+ + CS−)] for control and hM4Di-BLA→NAc^Vglut1 mice, indicating enhanced discriminative fear expression in Vglut1-hM4DiBLA→NAc mice (*p < 0.05, unpaired one-tailed t-test). Individual data points represent individual mice. Data are shown as mean ± SEM.

Reward conditioning is associated with altered intrinsic membrane properties of BLA→NAc neurons.

(A) Input resistance measured in Vglut1BLA→NAc projection neurons from naïve (blue) and reward-conditioned (pink) mice, (*p < 0.05, unpaired t test). (B) Current–voltage (I–V) relationship obtained under current-clamp conditions from Vglut1BLA→NAc projection neurons in naïve and conditioned mice. (C) Voltage–current relationship measured under voltage-clamp conditions from the same populations of neurons. Each point represents an individual recorded neuron from 3 mice/group. Error bars indicate mean ± SEM.