Fig. 4: Targeted optogenetic stimulation of ZI-RE dopaminergic projections enhances extinction recall.

A Experimental design: TH-CRE animals received intracranial injections of CRE-dependent control or ChR2 (ChannelRhodopsin2) virus in the ZI and after 4 weeks, were implanted with fiber optic cannula in the RE. After recovery from the implantation surgeries, animals were first habituated and then fear conditioned to tones using high shock intensities. 24 h later, animals were tested for fear generalization. Immediately after, animals went through an extinction session. The following day, animals were tested for extinction recall. B Outline of the high-intensity auditory fear conditioning protocol used in the study. On training day, both control and treatment groups of mice received CS+ tone presentations paired with 0.8 mA foot-shocks (high threat intensity) and unpaired CS− tone presentations. On testing day, freezing responses in both groups of animals were recorded for the CS+ and CS− tone presentations while laser stimulation occurred for the entire 30-s duration of each CS+ and CS− presentation. Immediately following the testing session and without removing animals from the chambers, animals received repeated presentations of CS+ tones as part of the extinction session with laser stimulation occurring for the entire 30-s duration of each CS+ presentation. One day later, fear responses of the animals to the CS+ tones were tested. C Animals injected with DIO-ChR2 virus in TH-CRE expressing A13 dopaminergic cells in the ZI and optic cannula in the RE receiving A13 dopaminergic projections (TH-DIO-ChR2-mCherry + stim + Vehicle) showed no significant differences in fear response to CS+ and CS− compared to animals that were infused with the DIO-mCherry virus (TH-DIO-mCherry + stim + Vehicle) in TH-CRE expressing A13 dopaminergic cells in the ZI and optic cannula in the RE receiving A13 dopaminergic projections. Pre-treatment with the DRD1 antagonist (SCH 23390, 0.1 mg/kg, i.p. in 0.9% saline Vehicle) did not affect freezing to the CS+ and CS− (TH-DIO-mCherry + stim + DRD1Antagonist, TH-DIO-ChR2-mCherry + stim + DRD1Antagonist). D Optogenetic activation of ZI-RE dopaminergic projections during extinction learning did not produce any significant differences in freezing responses between the four groups. E During extinction recall, animals that previously received optogenetic stimulation of ZI-RE dopaminergic projections (TH-DIO-ChR2-mCherry + stim + Vehicle) showed a significant decrease in fear response to CS+ compared to controls (TH-DIO-mCherry + stim + Vehicle). Importantly, pre-treatment with DRD1 antagonist blocked this enhancement of extinction recall (TH-DIO-ChR2-mCherry + stim + Vehicle vs TH-DIO-ChR2-mCherry + stim + DRD1 Antagonist) *p < 0.05, **p < 0.01. Data represented as Mean ± SEM.