C57BL/6 mice were purchased from CLEA Japan (Tokyo, Japan). The generation of D1R knockdown (KD) mice (D1R homozygous knockout/Tet/Off system-based compound-transgenic mice) was performed following previously published protocols (Chiken et al., 2015; Okubo et al., 2018; Saito et al., 2020). Only male mice were used in the contextual and cued fear conditioning tests.

Mice were maintained under a 12 h light/dark cycle (lights on at 7:00 AM), with ad libitum access to food and water in specific-pathogen-free conditions. All experiments were performed in accordance with the guidelines of the National Institutes of Health and the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan, and in compliance with the protocol that was reviewed by the Institutional Animal Care and Use Committee and approved by the President of Niigata University (Permit Number: SA00954) as previously described (Saito et al., 2020).

In D1RKD mice, 2.0 mg/mL doxycycline (Dox; Sigma Aldrich, United States) was used to knock down D1R expression, as previously described (Chiken et al., 2015; Okubo et al., 2018; Saito et al., 2020). For mice treated with Dox, Dox was administered via drinking water containing 5% sucrose for 4 weeks prior to behavioral tests and until the completion of the conditioning experiment (see section “Contextual and Auditory Cued Fear Conditioning Test,” Figures 1A, 2A), after which all mice received drinking water without Dox for the remainder period. Mice that were not treated with Dox received Dox-free water for the entire duration of the experiment. After termination of Dox administration, the expression level of D1Rs recovered to the same level as that before Dox administration (Chiken et al., 2015).

In the contextual and auditory cued fear conditioning test, D1RKD mice were assigned to D1RKD Dox (−) and D1RKD Dox (+) groups. D1RKD Dox (−) mice were used with ten mice per group. D1RKD Dox (+) were used with six and eight mice in recent and remote phase tests, respectively; six and ten wild-type (WT) mice were used as the control group in recent and remote phase tests, respectively.

In the immunohistochemical analysis, D1RKD mice were assigned to the following four groups: D1RKD Dox (−) Stimulation (−) (n = 3), D1RKD Dox (−) Stimulation (+) (n = 3), D1RKD Dox (+) Stimulation (−) (n = 3), and D1RKD Dox (+) Stimulation (+) (n = 3); two groups of WT mice, Stimulation (−) (n = 3) and Stimulation (+) (n = 3), were used as the control groups.

In the immunoblot analysis, D1RKD mice were assigned to the following six groups: D1RKD Dox (−) Stimulation (−) (n = 3), D1RKD Dox (−) Stimulation (+) (n = 3), D1RKD Dox (+) Stimulation (−) (n = 3), D1RKD Dox (+) Stimulation (+) (n = 3), D1RKD Dox (+→−) Stimulation (−) (n = 3), D1RKD Dox (+→−) Stimulation (+) (n = 3); two groups of WT mice, Stimulation (−) (n = 3) and Stimulation (+) (n = 3), were used as control groups.

A computer-controlled fear conditioning system was used for contextual and auditory cued fear conditioning tests (O’Hara & Co., Japan). A clear chamber (16 × 14 × 10 cm) for conditioning and contextual tests was set in a sound-insulating box (30 lux) with white walls. A white chamber (17 × 10 × 10 cm) for auditory cued tests was placed in a sound-insulating box (30 lux) with black walls. Mice were subjected to a conditioning session for 5 min on Day 1, a contextual test session for 5 min on Day 2 or Day 29, and an auditory cued test session for 5 min on Day 3 or Day 30.

In the conditioning session, a tone (65 dB, white noise) was presented for 20 s, and a mild footshock (0.3 mA, 2 s) was presented in the last 2 s through a floor grid. This process was performed three times for 5 min each. The mice were returned to their home cages 60 s after shock termination. Before the initial tone and footshock presentation and in the contextual test session, mice were placed back into a clear chamber used in the conditioning session for 5 min without footshock or tone stimulus. In the auditory cued test session, the mice were placed in a white chamber. A tone stimulus was presented for 2 min after 2 min of placing the mice in the white chamber. The behaviors of the mice were monitored using a CCD camera, and freezing behaviors were analyzed using Time FZ2 software (O’Hara & Co., Tokyo, Japan).

Footshock stimulation was carried out in a fear conditioning system or a step-through-type apparatus, comprising light and dark compartments separated by a removable door for the passive avoidance test (O’Hara & Co., Japan). In the fear conditioning system, each mouse was placed in the same way as in the fear conditioning tests. In the passive avoidance apparatus, each mouse was placed in the dark compartment and received a 2 s 0.3 mA electric footshock. Brain samples were collected 1 h after the footshock stimulus was delivered.

D1RKD mice and WT mice were anesthetized with a mixture of medetomidine hydrochloride [0.75 mg/kg body weight (BW)], midazolam (4 mg/kg BW), and butorphanol tartrate (5 mg/kg BW). The mice were then perfused with phosphate-buffered saline (PBS, pH 7.4) and 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4) through the left ventricle. The brains were removed, post-fixed overnight in 4% paraformaldehyde in 0.1 M PB (pH 7.4), and equilibrated overnight with 30% sucrose in 0.1 M PB (pH 7.4) at 4°C. The brains were embedded in OCT compound (Sakura Finetek, Japan), frozen over isopropanol (FUJIFILM Wako Pure Chemical Corporation, Japan) in liquid nitrogen, and cut on a cryostat CM1950 (Leica, Germany) into 40 μm thick coronal sections. Free-floating sections were treated with 3% H₂O₂ and 10% methanol in PB (pH 7.4) for 10 min and then blocked with 5% Blocking One (Nacalai Tesque) in 0.1% Triton in PBS (PBST) for 1 h after washing with PBS. Sections were incubated at 4°C with a polyclonal rabbit anti c-Fos antibody (1: 600, Cell Signaling) in blocking solution overnight, washed with PBST, and then incubated with biotinylated goat anti rabbit IgG (1: 300, Jackson Laboratories, United States) for 2 h. Thereafter, sections were incubated for 1 h with Vectastain Elite ABC Kit (Vector Laboratories, United States), washed with PBST, and then incubated with SIGMAFAST 3,3’-diaminobenzidine (DAB) (Sigma Aldrich, United States). After washing with distilled water, sections were mounted on glass slides, counterstained with 0.5% methyl green (FUJIFILM Wako Pure Chemical Corporation, Japan), and washed with distilled water. Finally, immunostained sections on mounted slides were fixed by serial dehydration in alcohol and lemosol and mounted using Softmount (FUJIFILM Wako Pure Chemical Corporation, Japan). Images of immunostained sections were acquired using a BZ-9000 microscope (Keyence, Japan) with BZ-II Analyzer software (Keyence, Japan). Immunoreactive cells were counted bilaterally in three sections, each from three mice by an experimenter blinded to the treatment conditions.

Regions in the brain of mice examined by immunohistochemistry are shown in Figure 3, and c-Fos positive cells were counted within the red rectangles. Brain sections including hippocampus and amygdala (Figure 3A; bregma −1.34 to −2.06 mm), DMS (Figure 3B; bregma −0.10 to 0.98 mm), and prelimbic region of mPFC (Figure 3C; bregma 1.70 to 1.98 mm) were analyzed. To examine each region of the hippocampus in detail, the number of c-Fos positive cells within the rectangles (300 × 300 μm) in the CA1, CA3, and dentate gyrus (DG) regions were counted separately. The number of c-Fos positive cells in the BA, DMS, and mPFC were counted within the rectangles (500 × 500 μm, 300 × 300 μm, and 300 × 300 μm, respectively), as shown in Figures 3A–C, respectively.

D1RKD mice and WT mice were sacrificed by cervical dislocation, and their brains were removed. The brains were sliced coronally into 1 mm thick sections. Regions in the brains of mice used for immunoblotting are shown in Figure 3. However, in contrast to immunohistochemistry, for immunoblotting, the entire hippocampus was used, including both the dorsal and ventral sides (Figure 3A). In addition, the entire striatum, including the DMS and the entire amygdala, including the BA was used (Figures 3A,B). Hippocampal tissues containing CA1, CA3, and DG were collected from the sections of three mice. Striatum and amygdala tissues were obtained from the sections of three mice. Each tissue was homogenized in phase-transfer surfactant buffer (PTS, 12 mM sodium deoxycholate, 12 mM sodium N-dodecanoylsarcosinate, and 200 mM triethylammonium bicarbonate) with cOmplete EDTA-free protease inhibitor (Sigma Aldrich, United Kingdom), PhosStop (Sigma Aldrich, United Kingdom), and 1 mM EDTA. Homogenized proteins were quantified using a BCA kit (Wako, Japan). Homogenates were solubilized in sample buffer (125 mM Tris–HCl, pH 7.4, 3.3% glycerol, 2% SDS, and 50 mM DTT) and heated at 90°C for 5 min. The proteins were then separated on 5 – 20% polyacrylamide gel (ATTO, Japan) loaded at 5 μg in SDS sample buffer at 15 mA for 90 min. These gels were blotted onto PVDF membranes (Immobilon-P, Merck Millipore) in blotting buffer (192 mM glycine, 0.037% SDS, 20% methanol, 100 mM Tris–HCl, pH 8.8) at 20 V for 60 min, and then blocked with 5% non-fat milk in TBST (150 mM NaCl, 0.2% Tween 20, 20 mM Tris–HCl, pH 7.5) for 30 min at room temperature. These membranes were incubated with rabbit anti c-Fos antibody (1: 1,000, Cell Signaling), mouse anti actin antibody (1: 1,000, BD Biosciences, United States), rabbit anti SNAP-25 antibody (1 μg/ml) (Yamamori et al., 2011), or rabbit anti Phospho-SNAP-25 (Ser187) antibody (1: 500) (Iida et al., 2013) in 1% non-fat milk with TBST at 4°C overnight, washed with TBST, and then incubated with peroxidase-conjugated donkey anti-rabbit IgG (H + L) (1: 2,000, Jackson Laboratories, United States) or peroxidase-conjugated donkey anti-mouse IgG (H + L) (1: 1,000, Jackson Laboratories, United States) for 1 h. Immunoreacted samples were visualized using a chemiluminescent reagent (Chemi-Lumi One, Nacalai Tesque or Immobilon Western Chemiluminescent HRP, Sigma Aldrich), and images were acquired using a luminescence image analyzer (LAS 4000, GE Healthcare, Sweden) with ImageQuant TL software (GE Healthcare, Sweden).

All data were analyzed using Origin 2021 (OriginLab, United States). Data from the contextual and auditory cued fear conditioning tests and cell counting data from immunohistochemistry were analyzed using the Mann-Whitney test. Immunoblot data were analyzed using the two sample t test. Significance was set at p < 0.05.

The effect of suppressing D1R expression on fear memory formation was examined using a contextual and auditory cued fear conditioning test in the recent and remote phase (Figure 1A). First, recent phase examinations found significantly longer freezing responses in both the context test (Day 2) and auditory cued test (Day 3) than those before conditioning. There was no significant difference between the three groups on both the context test (Day 2) and auditory cued test (Day 3) (Figure 1B). Next, the remote phase contextual and auditory cued fear conditioning tests were conducted using different mice from those used in the recent phase tests. In the remote phase context tests (Day 29), D1RKD Dox (+) mice showed almost no freezing response, similar to before conditioning (Day 1). The freezing responses of D1RKD Dox (+) mice were clearly different from those of the other two groups (D1RKD Dox (−) and WT), while the freezing responses in the remote phase context tests (Day 29) of D1RKD Dox (−) and WT mice were increased and similar to those seen in the recent phase context tests (Day 2). On the other hand, D1RKD Dox (+) mice showed a significantly increased percentage of time spent freezing in the auditory cued tests (Day 30), which was equivalent to WT and D1RKD Dox (−) mice, albeit with some amount of variation (Figure 1C). Furthermore, D1RKD Dox (−) mice overexpressing D1Rs showed a significantly increased percentage of time spent freezing compared to WT mice in the auditory cued tests [Day 3 (p < 0.05), Day 30 (p < 0.05)].

In addition, the effects of recovery of D1R expression were examined using the same mice as those in the remote phase contextual and auditory cued fear conditioning tests (Figure 2A). In the first test phase in the context tests (Day 29), D1RKD Dox (+) mice showed almost no freezing response, similar to before conditioning (Day 1) (Figure 2B). In the second test phase, following footshock conditioning on Day 1, D1RKD Dox (+) mice showed a significantly increased percentage of freezing on the context tests (Day 29). On the other hand, in the auditory cued fear conditioning test, both D1RKD mice with suppressed D1R expression in the first test phase and the same mice with recovering D1R expression in the second phase showed freezing behavior. These results using the same D1RKD mice were consistent with those obtained using different D1RKD mice (Figure 2A).

Representative immunohistochemical staining images of the hippocampus are shown in Figure 4A. Hippocampal CA1, CA3, and DG regions in Figure 4 were enlarged and shown in Supplementary Figures 1A–C, respectively. Distributions of c-Fos positive cells in the hippocampus of WT and D1RKD Dox (−) mice with footshock stimulation by a fear conditioning (FC) or a passive avoidance (PA) apparatus were compared with those of the WT and D1RKD Dox (−) mice without stimulation. In the case of PA footshock stimulation, there was an increase in the number of c-Fos positive cells in a large area of the hippocampi of WT and D1RKD Dox (−) mice with footshock stimulation. In contrast, c-Fos positive cells were hardly observed in either case of D1RKD Dox (+) mice with or without stimulation (Figure 4A). In the case of FC footshock stimulation, the number of c-Fos-positive cells increased in a large area of the hippocampi of footshock-stimulated WT mice. In contrast, c-Fos-positive cells were not often observed in either D1RKD Dox (−) and D1RKD Dox (+) mice, with or without stimulation (Figure 4A). In both cases of PA and FC footshock stimulation, when the number of c-Fos positive cells was counted in all the CA1, CA3, and DG regions, there was a significant increase in the two groups (WT and D1RKD Dox (−) mice) when comparing the stimulated and unstimulated groups. However, the number of positive cells D1RKD Dox (−) mice was lower in FC than in PA. Moreover, in the case of PA apparatus, there was a little increase in the hippocampal CA1, CA3, and DG regions of the D1RKD Dox (+) mice group when comparing the stimulated and unstimulated groups (Figure 4B). In the case of FC footshock stimulation, the number of positive cells in the hippocampal CA1 and CA3 regions was slightly increased, but that in the DG region was almost unchanged (Figure 4B). In addition, the distributions of c-Fos positive cells in the DMS of WT, D1RKD Dox (−), and D1RKD Dox (+) mice with and without stimulation were found to be similar to those observed in the hippocampus as described above (Figure 5A). When the number of c-Fos positive cells was counted in DMS, there was a significant increase in the both groups (WT and D1RKD Dox (−) mice) when comparing the stimulated and unstimulated groups. However, there was a little increase in the DMS or hippocampus of the D1RKD Dox (+) mice group when comparing the stimulated and unstimulated groups (Figure 5B). In contrast, an apparent increase in the number of c-Fos positive cells was observed in the mPFC and BA in the mice of the three groups [WT, D1RKD Dox (−), and D1RKD Dox (+)] that received stimulation (Figures 6A, 7A). Furthermore, the number of c-Fos positive cells in mice with stimulation was significantly different from that in mice without stimulation in the three groups (p < 0.001) (Figures 6B, 7B).

Representative immunoblotting images are shown in Figure 8. First, in the case of FC footshock stimulation, c-Fos protein levels were greatly increased in both groups (WT, D1RKD Dox (−) mice), but the increase in c-Fos protein levels in D1RKD Dox (+) mice was quite small compared with those in WT and D1RKD Dox (−) mice in the hippocampus and striatum. Second, in the case of PA footshock stimulation, c-Fos protein levels were significantly increased in D1RKD Dox (−) mice, but those in D1RKD Dox (+) mice were relatively small (Figure 8C). These results were consistent with those of immunohistochemical analysis, except for the increase in the hippocampal CA1 region in the case of FC apparatus. Furthermore, c-Fos protein levels were also examined in D1RKD Dox (+→−) mice in which D1R expression was restored after termination of Dox administration. c-Fos protein levels tended to be similar to those in D1RKD Dox (−) mice before Dox administration. This result indicates that the restoration of D1R expression following suppression led to the recovery of the response to stimulation with respect to the induction of c-Fos expression.

In contrast, in the amygdala, the amount of c-Fos protein was greatly increased by stimulation in all four groups (WT, D1RKD Dox (−), D1RKD Dox (+), and D1RKD Dox (+→−) mice), and also these results were consistent with the results of immunohistochemical analysis, in the case of FC footshock stimulation. In the case of PA footshock stimulation, c-Fos protein levels increased slightly more than in the case of FC footshock stimulation, but the same tendency as that of FC stimulation was observed (Figure 8C). The amount of SNAP-25 protein was approximately the same in all four groups (WT, D1RKD Dox (−), D1RKD Dox (+), and D1RKD Dox (+→−) mice).

Next, in the case of FC footschock stimulation, the amount of phosphorylated SNAP-25 at Ser187 tended to decrease with stimulation in D1RKD Dox (−), D1RKD Dox (+→−), and D1RKD Dox (+) mice in all observed regions of the hippocampus, striatum, and amygdala. In addition, in the case of PA footshock stimulation, the amount of phosphorylated SNAP-25 at Ser187 significantly increased in D1RKD Dox (−), D1RKD Dox (+→−) mice in region of the striatum following stimulation. In contrast, in the hippocampus and amygdala, there were no significant changes. Interestingly, there was a large increase in the amount of phosphorylated SNAP-25 at Ser187 not only after stimulation but also before stimulation of the D1RKD Dox (+) mice compared to that in WT mice without stimulation. This result was prominent in regions of the striatum and amygdala (Figures 8A,B,D).