Supplementary Figure 2. Effects of the D1 Receptor Agonist SKF38393 During Fear Extinction

Supplementary Figure 1.Effect of Gq-DREADD-induced activation of substantia nigra (SN) dopamine (DA) neurons on cFos immunoreactivity following fear renewal. Rats were exposed to the extinguished conditioned stimulus in either the familiar extinction context (Same) or a novel context (Different) and then cFos immunoreactivity was quantified in the infralimbic (IL) and prelimbic (PL) regions of the prefrontal cortex, the basolateral (BLA) and central (CeA) nuclei of the amygdala, and dentate gyrus (DG), CA3, CA2, and CA1 regions of the hippocampus. (A) Coronal diagrams modified from the atlas of Paxinos and Watson (1998) showing regions quantified. Representative photomicrographs of the CA1 region from a (B) Vehicle Same rat and (C) SN CNO Different rat. (D) Rats re-exposed to the extinguished CS in the Same context in which extinction was learned demonstrated higher levels of cFos in the IL than rats re-exposed to the extinguished CS in the Different context, although the main effect of context just missed significance (F(1,22) = 3.876, p = 0.06; Figure S1B). SN DA activation had no significant impact on cFos expression in the IL, suggesting that the IL is unlikely to be the site of action mediating the effects of SN DA activation. No group differences in cFos expression were observed in the PL. (E)Consistent with the levels of freezing observed in the Different context, cFos expression in the CeA was significantly higher in rats placed into the Different context than those placed into the Same context (F(1,22) = 5.784, p = 0.02). Interestingly, though, cFos expression in the CeA of rats whose SN DA neurons were activated during fear extinction was identical between contexts. No significant differences were observed in the BLA, CA3, or CA2. In the DG; however, cFos expression was higher in rats re-exposed to the extinguished CS in the Same context compared to the Different context (F(1, 21) = 4.131, p = 0.05). In the CA1, highercFos expression was observed in rats whose SN DA neurons were activated during fear extinction compared to rats in other groups, regardless of the context in which rats were re-exposed to the extinguished CS (F(2,21) = 3.566, p = 0.04. N = 4-7 per group. Data presented represent means ± SEM. *p < 0.05 relative to Same counterparts;  p< 0.05 main effect of context; # p < 0.05 SN CNO relative to Vehicle and Off-Target CNO groups regardless of context.

Supplementary Figure 2. Effects of the D1 receptor agonist SKF38393 during fear extinction on drug-free extinction memory.(A) Experimental design.Saline (n=8) or SKF38393 (n=8) were microinjected into the DS 10 min prior to fear extinction day 1. (B) Coronal sections modified from the Paxinos and Watson (1998) rat brain atlas showing the location of cannula placements. (C)All rats acquired fear conditioning throughout auditory fear conditioning (Main effect of time: F(3,42) = 25.201, p < 0.0001). (H) The following day, all rats acquired within-session fear extinction (Main effect of time: F(5,70) = 72.155, p < 0.0001) regardless of drug treatment.(I) When tested the following day drug-free, D1 receptor activation during extinction failed to enhance fear extinction memory. Data presented represent means ± SEM.

Supplementary Table 1

Table S2. Cage crossings. Given that SN DA neurons and D1 receptors in the DS are closely associated with movement, cage crossings were quantified during the 3-min pre-freezing period, prior to the first CS exposure, on extinction day 1 and day 2 as a measure of locomotor activity. Data presented represent means ± SEM.

Supplementary Materials and Methods

Immunohistochemistry

Brains from TH-Cre rats not used for FISH were removed 90 min after fear renewal testing and prepared as previously described (Herrera et al, 2016; Lloyd et al, 2017). Thirty-five m coronal sections through the PFC (3.2 to 1.7 mm Bregma), AMG / hippocampus (-1.8 to -4.3 mm Bregma) or midbrain (-4.5 to -7.0 mm Bregma) were incubated in rabbit anti-cFos(1:3000; Santa Cruz Biotechnology, Inc. sc-52; Dallas, TX) or mouse anti-TH (1:1000; Immunostar; Hudson, WI). cFos was visualized using nickel-enhanced 3,3’-diaminobenzidine and TH was visualized with fluorescent Donkey anti-Mouse IgG (H&L) DyLight 650 Conjugate (1:2000; ImmunoReagents, Inc; Raleigh, NC). Images of serial sections spaced 200 µm apart were captured at 200X magnification on an Olympus BX53. Cells expressing cFos protein within the counting frame (1.48 x 105 m) were counted by multiple experimenters blind to treatment conditions (6-10 hemispheres per region per animal). Single TH+ (green; A488), mCherry+ (red; A568), or double-labeled cells were quantified from 6 hemispheres of 5 representative rats using a Zeiss Axio Observer Z1 laser scanning confocal microscope (Zeiss Microscopy).

Behavior

All contexts were contained within sound-attenuating cabinets. Context A consisted of a custom Plexiglass conditioning chamber (20 W x 10 D x 12 H cm; Context A) with a shock grid floor (Coulbourn Instruments, Allentown, PA). A fan provided ventilation and background noise, dim white light illuminated the room, and chambers were cleaned with water. Context B was either a Plexiglas rectangular chamber with a smooth floor (15”W x 15”D x 20”H) or a Plexiglas triangular chamber with a textured floor (15”D sides x 20”H). Rectangular and triangular Context B inner chambers were counterbalanced so half the rats were exposed to fear extinction in each chamber. Context B contained vanilla scent, bright white box lights, and no background noise. Context B was cleaned with 10% EtOH between rats. For fear renewal testing, rats were re-exposed to the auditory CS in either the familiar Context B (Same) or assigned to a novel Context C (Different). Context C consisted of a novel inner chamber (square or triangle), raspberry scent, red box lights, and background noise from a different fan than that used in context A. Context C was cleaned with 1% acetic acid between tests. Rats were transported to Context A in their home cages and to Context B and C in their assigned Plexiglass inner chambers. Rats explored their assigned context for 3 min prior to the first CS, and remained in that context for 1 min after the last CS. Coulbourn shock scramblers and speakers were controlled with EthoVision XT (Noldus, Leesburg, VA). All behavioral tests were recorded with overhead cameras, and freezing was scored by multiple experimenters blind to experimental conditions and by automated behavioral analyses software (NoldusEthovision XT). Because regularly-scheduled ITIs can become a part of the CS, freezing during each 10s CS and subsequent 60s ITI were combined and expressed as freezing during a trial.