Figure 5. DA via DRD1 mediates presynaptic depression between ITC clusters.

(A) Confocal images of the amygdala showing ChR2-YFP-expressing neurons in the dorsomedial-intercalated cell (dm-ITC) cluster and their efferents targeting the ventromedial-intercalated cell (vm-ITC) cluster after injection of AAV-DIO-ChR2-YFP in FoxP2-Cre mice. Left: Overview of the amygdala. Right: Details of the dm-ITC injection site with YFP+ cells (top) and vm-ITC recording site with YFP+ fibers (bottom). Scale bars: 200 and 20 µm. (B) Time course of changes in inhibitory postsynaptic current (IPSC) amplitude upon bath application of dopamine (DA, 30 µM, 5 min) in the dm-ITC→vm-ITC pathway. DA decreased IPSC amplitude, which returned to near baseline levels upon washout. (C) Top: Representative IPSC traces recorded at 0 mV from the baseline period and during DA application (paired pulse interval 100 ms). Scale bars: 50 pA, 50 ms. Bottom: Relative change of IPSC amplitude and paired pulse ratio (PPR) during DA application (n=6 cells from three animals, amplitude 41.53 ± 7.25%, paired t-test, **p=0.003; PPR 180.00 ± 25.01%, paired t-test, *p=0.031). (D and G) Left: Schematics of experimental approach with infection, recording, and light stimulation sites. Right: Representative traces recorded at baseline (black) and during agonist application (colored). Dihydrexidine (DH, 10 µM), a DRD1 agonist, is shown in red, Quinpirole (Quinp, 1 µM), a DRD2 agonist, is shown in blue (paired pulse interval: 100 ms). Scale bars 50 pA, 50 ms. (E and H) Comparison of relative changes in IPSC amplitude during agonist application in the dm-ITC→vm-ITC pathway (E) and l-ITC→dm-ITC pathway (H). (E) DH (red, n=6 cells from four animals) suppressed IPSCs (amplitude 46.69 ± 8.14%, **p=0.010, paired t-test), whereas Quinp (blue, n=6 cells from three animals) had no effect (amplitude 98.45 ± 5.37, p=0.438, paired t-test). Between-group analysis revealed a significant drug effect on the dm-ITC→vm-ITC amplitude (one-way ANOVA, F(2, 15)=20.107, p<0.001) with Quinp differing from DA and DH (###p<0.001 each, Bonferroni post-hoc tests). (H) DH (red, n=9 cells from five animals) strongly suppressed IPSCs (amplitude 47.16 ± 6.67%, ***p<0.001, paired t-test), whereas Quinp (blue, n=7 cells from three animals) had a minor effect (amplitude 92.38 ± 1.63%, **p=0.006). Between-group analysis revealed a significant drug effect on the l-ITC→dm-ITC amplitude (one-way ANOVA, F(2, 19)=17.394, p<0.001) with Quinp differing from DA and DH (###p<0.001 each, Bonferroni post-hoc tests). (F and I) Comparison of relative change of PPR during drug application in the dm-ITC→vm-ITC pathway and l-ITC→dm-ITC pathway. (F) DH (red, n=6) increased PPR (181.37 ± 25.38%, *p=0.035, paired t-test), whereas Quinp (blue, n=6) had no significant effect on PPR (123.97 ± 11.25%, p=0.127, paired t-test). Between-group analysis revealed no significant drug effect on the dm-ITC→vm-ITC PPR (one-way ANOVA, F(2, 15)=2.304, p=0.134). (I) DH (red, n=9) increased PPR (158.46 ± 18.52%, **p=0.005, paired t-test), whereas Quinp (blue, n=7) had no effect on PPR (105.02 ± 4.98%, p=0.385, paired t-test). Between-group analysis revealed a significant drug effect on the l-ITC→dm-ITC PPR (one-way ANOVA, F(2, 19)=3.804, #p=0.041). (J and K) Combined data from both pathways show a significant correlation between change in PPR and change in amplitude upon application of DA (J: Pearson correlation, r=−0.78, p=0.003, n=12) and upon application of DH (K: Pearson correlation, r=−0.55, p=0.035, n=15).

Figure 5—figure supplement 1. An l-ITC→dm-ITC inhibitory pathway that is modulated by DA via a presynaptic mechanism.

(A) Experimental scheme. AAV-DIO-ChR2-YFP was injected into the amygdala of FoxP2-Cre mice to specifically infect the lateral intercalated cell (l-ITC) cluster, and optically evoked postsynaptic currents (PSCs) were recorded in dorsomedial-intercalated cells (dm-ITCs). (B) PSCs recorded in a dm-ITC at different holding potentials (−70 mV to +40 mV) with a reversal potential of −41.44 mV. Application of the GABA_A channel blocker picrotoxin (PTX) 100 µM) abolished the PSCs. Scale bars 200 pA, 20 ms. (C) Box plots and individual latencies (circles) for IPSCs evoked between different ITC clusters. Synaptic latencies were similar for the l-ITC→dm-ITC pathway (2.07 ± 0.10 ms, n=22 cells from 12 animals) and the dm-ITC→vm-ITC pathway (2.23 ± 0.09 ms, n=18 cells from 10 animals, unpaired t-test, p=0.268). (D) Confocal images showing ChR2-YFP-expressing neurons in the l-ITC cluster and their efferents targeting the dm-ITC cluster. Left: Overview of the amygdala. Right: Close-up of the injection sites with YFP+ cells in the l-ITC (top) and recording sites with YFP+ fibers in the dm-ITC cluster (bottom). Scale bars 200 and 20 µm. (E) Time course of changes in IPSC amplitude upon bath application of dopamine (DA, 30 µM, 5 min) in the l-ITC→dm-ITC pathway. DA decreased IPSC amplitude, which returned to near baseline levels upon washout. (F) Top: Representative IPSC traces recorded at 0 mV from the baseline period and during DA application (paired pulse interval 100 ms). Scale bars 50 pA, 50 ms. Bottom: Relative change of IPSC amplitude and paired pulse ratio (PPR) during DA application (n=6 cells from three animals, amplitude 48.05 ± 8.09%, paired t-test, **p=0.006; PPR 165.72 ± 20.46%, paired t-test *p=0.019).