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. 2021 May 24;10:e63708. doi: 10.7554/eLife.63708

Figure 6. Early extinction enhances GABA release from midbrain terminals and DA-mediated depression of the dm-ITC→vm-ITC pathway.

(A) Experimental scheme: To investigate the VTA/SNC→dm-ITC pathway, ChR2 was transduced in dopaminergic midbrain neurons of DAT-Cre mice. To investigate dopamine (DA) modulation of the dm-ITC→vm-ITC pathway, ChR2 was transduced into the dorsomedial-intercalated cell (dm-ITC) cluster of FoxP2-Cre mice. The early extinction group (E-Ext) underwent fear conditioning on day 1 (5 conditioned stimulus-unconditioned stimulus (CS-US) pairings) and early extinction training on day 2 (16 CS presentations). The CS-only group received only CS presentations. (B) Left: Example traces of light-evoked postsynaptic currents (PSCs) by dopaminergic fiber stimulation recorded in dm-ITCs at −70, 0, and 40 mV from CS-only (grey traces) or E-Ext animals (blue traces). Scale bars: 50 pA, 50 ms. (C) Plot of PSC reversal potentials in individual dm-ITCs (dots) and average (red lines) from CS-only and E-Ext groups. Erev was significantly lower in the E-Ext (−45.09 ± 1.76 mV, n=11 cells from six animals) vs. the CS-only group (−34.06 ± 5.11 mV, n=10 cells from four animals, *p=0.047, paired t-test). (D) Summary graph comparing the type of fast PSCs in dm-ITCs recorded from CS-only and E-Ext animals (CS-only, n=10: GABA 30%, mixed 60%, Glu 10%; E-Ext, n=11: GABA 82%, mixed 18%). PSC types were significantly different between groups (Fisher’s exact test = 5.68, *p=0.041). (E) Example traces of light-evoked inhibitory postsynaptic currents (IPSCs) recorded in a ventromedial-intercalated cell (vm-ITC) at 0 mV upon paired pulse stimulation (100 ms interstimulus interval) of the dm-ITC→vm-ITC pathway from CS-only (grey traces) or E-Ext (blue traces) animals before (solid) and during DA application (dotted). Scale bars 50 pA, 50 ms. (F) Time course of changes in IPSC amplitude upon bath application of DA (30 µM, 5 min) in dm-ITC→vm-ITC pathway in CS-only and E-Ext groups. Two-way ANOVA (1–9 min) revealed significant changes for time, F(8)=37.903, p<0.001, and group, F(1)=8.229, p=0.005, but no significant interaction, F(8, 144)=0.521, p=0.839. (G) Significant changes of IPSC amplitude (paired t-tests: CS-only, **p=0.002; E-Ext, ***p<0.001) and paired pulse ratio (PPR) (paired t-tests: CS-only, *p=0.040; E-Ext, **p=0.003) 4–5 min after DA application in both groups. IPSC amplitude was more depressed in neurons recorded from E-Ext vs. CS-only animals (32.42 ± 3.16%, n=11 cells from six animals, vs. 46.75 ± 6.14%, n=7 cells from four animals, unpaired t-test, #p=0.036). The PPR increase was larger in neurons recorded from E-Ext vs. CS-only animals (310.63 ± 42.64%, n=11, vs. 139.68 ± 15.43%, n=7, unpaired t-test, ##p=0.007).

Figure 6—source data 1. Data Figure 6C–D.
Figure 6—source data 2. Data Figure 6F.
Figure 6—source data 3. Data Figure 6G.

Figure 6.

Figure 6—figure supplement 1. Behavioral data from DAT-Cre mice used for ex vivo recordings and viral injection sites.

Figure 6—figure supplement 1.

(A) Experimental strategy for targeting of dopaminergic midbrain neurons by injecting DAT-Cre mice with AAV-DIO-ChR2-YFP. (B) Freezing levels for behavioral paradigms shown in Figure 6A in virally transduced DAT-Cre mice used for recordings. A group of mice was subjected to only conditioned stimulus (CS) presentations (CS-only, grey, n=4 mice), and the other group was subjected to fear conditioning on day 1 and early extinction training with 16 CS presentations (E-Ext, blue, n=6 mice). Repeated-measures ANOVA, F(3, 15)=1.382, p=0.287, reveals no significant differences between time points for E-Ext. (C) Brain atlas overlays of sites showing the distribution of ChR2-YFP-labeled neuronal cell bodies in the dopaminergic midbrain of DAT-Cre mice in which fast postsynaptic currents (PSCs) were recorded. Top: CS-only (n=4 mice); bottom: E-Ext (n=6 mice). Bregma levels are indicated at the top.
Figure 6—figure supplement 1—source data 1. Data Figure 6—figure supplement 1B.
Figure 6—figure supplement 2. Behavioral data from mice trained with a long extinction protocol.

Figure 6—figure supplement 2.

Another group of DAT-Cre mice was subjected to extinction training with 25 conditioned stimulus (CS) presentations on days 2 and 3, and extinction recall by four CS presentations on day 4 (n=10 mice). On days 2 and 3, freezing was averaged across four CSs for the first five time points and five CSs for the last one. Across days 2–4, a repeated-measures ANOVA, F(3, 27)=6.467, p=0.002, reveals significant differences between time points; Bonferroni-corrected pairwise comparisons: day 2 (CS1–4) vs. day 2 (CS21–25), *p=0.039; day 2 (CS1–4) vs. day 3 (CS21-25) and vs. day 4 (CS1-4), *p=0.015 and *p=0.018, respectively. For within-session extinction on day 2, a repeated-measures ANOVA, F(5, 45)=8.14, p<0.001, reveals significant differences between CS blocks; Bonferroni-corrected pairwise comparisons: (CS1–4) vs. (CS17–20), #p=0.045.
Figure 6—figure supplement 2—source data 1. Data Figure 6—figure supplement 2.
Figure 6—figure supplement 3. Behavioral data from FoxP2-Cre mice used for ex vivo recordings.

Figure 6—figure supplement 3.

(A) Experimental strategy for targeting of dorsomedial-intercalated cell (dm-ITC) clusters by injecting FoxP2-Cre mice with AAV-DIO-ChR2-YFP. (B) Freezing levels of virally transduced FoxP2-Cre mice used for recordings (conditioned stimulus (CS)-only group, grey, n=4 mice; E-Ext group, blue, n=6 mice) are displayed as described in Figure 6—figure supplement 1. Repeated-measures ANOVA, F(3, 15)=0.647, p=0.597, reveals no significant differences between time points for E-Ext.
Figure 6—figure supplement 3—source data 1. Data Figure 6—figure supplement 3B.