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. Author manuscript; available in PMC: 2024 Aug 9.
Published in final edited form as: J Psychopharmacol. 2024 Jul 28;38(7):672–682. doi: 10.1177/02698811241261375

No evidence that post-training dopamine D2 receptor agonism affects fear generalization in male rats

Natalie Schroyens 1,2, Laura Vercammen 1,2,3,✉, Burcu Özcan 1, Victoria Aurora Ossorio Salazar 2,3, Jonas Zaman 4, Dimitri De Bundel 5, Tom Beckers 1,2,✉, Laura Luyten 1,2,✉
PMCID: PMC7616352  EMSID: EMS197949  PMID: 39068641

Abstract

Background

The neurotransmitter dopamine plays an important role in the processing of emotional memories, and prior research suggests that dopaminergic manipulations immediately after fear learning can affect the retention and generalization of acquired fear.

Aims

The current study focuses specifically on the role of dopamine D2 receptors (D2Rs) regarding fear generalization in adult, male Wistar rats, and aims to replicate previous findings in mice.

Methods

In a series of five experiments, D2R (ant)agonists were injected systemically, immediately after differential cued fear conditioning (CS+ followed by shock, CS- without shock). All five experiments involved the administration of the D2R agonist quinpirole at different doses versus saline (n = 12, 16 or 44 rats/group). Additionally, one of the studies administered the D2R antagonist raclopride (n = 12). One day later, freezing during the CS+ and CS- was assessed.

Results

We found no indications for an effect of quinpirole or raclopride on fear generalization during this drug-free test. Importantly, and contradicting earlier research in mice, the evidence for the absence of an effect of D2R agonist quinpirole (1 mg/kg) on fear generalization was substantial according to Bayesian analyses and was observed in a highly powered experiment (N = 87). We did find acute behavioral effects in line with the literature, for both quinpirole and raclopride in a locomotor activity test.

Conclusion

In contrast with prior studies in mice, we have obtained evidence against a preventative effect of post-training D2R agonist quinpirole administration on subsequent fear generalization in rats.

Keywords: differential cued fear conditioning, generalization, rats, dopamine, quinpirole, raclopride

1. Introduction

The neurotransmitter dopamine has been shown to play a role in the processing of emotional memories (Iemolo et al., 2015). For instance, genetic and pharmacological studies suggest that activation of dopaminergic D1 and D2 receptors (D1Rs and D2Rs) immediately after Pavlovian conditioning or inhibitory avoidance training is required for subsequent expression of acquired fear (Castellano et al., 1991; Fadok et al., 2009; Greba et al., 2001; LaLumiere et al., 2004; Manago et al., 2008). In addition, dopamine has been found to modulate long-term potentiation (Bissière et al., 2003; Frey et al., 1991; Lemon and Manahan-Vaughan, 2006; Rosenkranz and Grace, 2002; Rossato et al., 2009), a form of synaptic plasticity that is assumed to be essential for memory formation (Lisman and Grace, 2005). The focus of the present paper is on the role of dopaminergic D2R signaling after fear conditioning with two cues, differentially predicting danger (CS+) and the absence of danger (CS-), in subsequent fear generalization from the CS+ to the CS-.

Although most studies regarding the influence of dopamine on fear memory processing have focused on its effects on memory retention specifically, more recent studies have also investigated its role in generalization of fear towards safe or novel stimuli after contextual, cued, or differential cued fear conditioning (De Bundel et al., 2016; Jo et al., 2018; Jones et al., 2015; Sarinana et al., 2014; Zweifel et al., 2011). Investigating if and how dopaminergic manipulations during or after learning affect the retention and generalization of fear towards safe situations is highly relevant to understand the neurobiology of anxiety, given that such fear generalization is often found to be elevated in patients with anxiety disorders (Hermans et al., 2013; Lee et al., 2016).

Prior research indicated that knock-out mice lacking hippocampal D1Rs exhibit equal levels of freezing during exposure to a novel context as to a context that was previously paired with shock (Sarinana et al., 2014). In addition, using differential cued fear conditioning procedures in mice, it has been shown that fear generalization from the CS+ to CS- can be countered by optogenetic stimulation of dopaminergic neurons during conditioning (Jo et al., 2018) or by systemic injection of a D2R agonist immediately after conditioning (De Bundel et al., 2016). The latter study, by De Bundel and colleagues, found the dopaminergic effect to be bidirectional, with post-learning injection or intra-amygdala infusion of a D2R antagonist increasing the generalization from the CS+ to the CS- relative to the saline groups in these experiments. A PET imaging study in humans (Frick et al., 2021) found evidence consistent with these findings in mice. Likewise, recent rat studies (often with more elaborate behavioral procedures that also included reward cues, or using compound or contextual stimuli, rather than simple differential learning with a tone CS+ and CS- (Colucci et al., 2019; Moaddab and McDannald, 2021; Ng et al., 2018; Yau and McNally, 2022)) have provided accumulating evidence for the involvement of dopamine signaling in the discrimination between danger and the absence of danger. The pharmacological and recording techniques that were used in these rat studies did, however, not allow for conclusions regarding the specific involvement of D2 receptors.

These previous neurobiological studies have proposed a range of different mechanisms that may underlie the effect of dopamine on the spreading of fear towards safe situations. For example, it has been suggested that the involvement of dopamine in the encoding of novel perceptual information may lie at the basis of its effects on fear generalization (Sarinana et al., 2014). Alternatively, it has been proposed that the generalized fear observed in knockout mice with impaired dopaminergic signaling can be attributed to inadequate learning of CS- US associations due to aberrant salience detection (Zweifel et al., 2011) and the erratic assignment of negative valence to both the CS+ and CS- during learning (Jo et al., 2018). Yet another possible way in which dopamine may limit fear generalization from stimuli that predict danger to those that predict safety is via enhancement of the stabilization or ‘consolidation’ of the acquired fear memory (De Bundel et al., 2016). Related to this idea, it has been suggested that ensuring adequate stabilization of the fear memory trace may be required for the establishment of a sufficiently precise memory to prevent generalization to non-threatening situations (Robertson, 2018). Overall, dopamine’s effects on fear memory generalization have thus been attributed to several aspects of learning (i.e., encoding of perceptual information, association formation, differential learning), at different points in time (i.e., during versus shortly after learning), and via various hypothetical mechanisms (i.e., by coding salience or valence, or by enhancing memory stabilization).

Our ultimate goal was to study, in rats rather than mice, to what extent dopaminergic D2R signaling after differential fear learning plays a role in subsequent fear generalization and, if so, which aspect(s) of learning (perceptual, associative, and/or differential) would be involved. To this aim, we first embarked on a conceptual replication of published findings in mice by De Bundel and colleagues (2016), in which it was shown that systemic injection of a D2R agonist or antagonist immediately after differential cued fear conditioning can attenuate or augment subsequent fear generalization, respectively (i.e., decrease or increase fear responding to a safe cue in a new context). One advantage of administering dopaminergic drugs after learning rather than before is that it allows to distinguish between the importance of dopaminergic signaling after learning versus during. It may thus provide useful insight into the role of dopamine D2Rs during the memory consolidation phase specifically, without affecting encoding.

While most prior evidence in non-human animals regarding the role of dopamine D2Rs in attenuating or augmenting fear generalization has been obtained in mice, the translation of those findings to rats remains unclear. Despite the many similarities between both species, rats and mice are characterized by important differences in functional brain anatomy and physiology, including neurochemistry (Ellenbroek and Youn, 2016). Rats are an important model organism for behavioral neuroscience, in particular in the study of fear learning and interventions to influence fear memory specificity. Moreover, establishing the generalizability of the findings in mice to rats may also inform us about their potential relevance for other organisms, including humans. Exactly copying behavioral procedures from mice to rats is, however, rarely advisable, so we slightly adapted the fear learning tasks from (De Bundel et al., 2016) in order to make them more appropriate for rats and less sensitive to order and context effects.

We describe a series of five experiments (Exp. 1-5) that investigated the behavioral effects of post-training dopaminergic drug injection on subsequent fear generalization in rats. These experiments were preceded by a series of protocol optimization studies (reported as Exp. A-C) that provided insight into the effects of shock intensity and testing order on fear responding to the CS+ and CS-. In a last experiment (Exp. 6), the acute effects on locomotor activity of the drugs used throughout our experiments was evaluated (i.e., 0.3 mg/kg raclopride and 0.05, 1, and 5 mg/kg quinpirole). Details regarding Experiments A-B-C and Experiment 6 can be found in the Supplement.

2. Experimental Procedures

2.1. Protocol registration

All study protocols and statistical analyses were preregistered on the Open Science Framework (OSF) at https://osf.io/kzcyt.

2.3. Subjects

Male Wistar rats (Janvier Labs, Le Genest-Saint-Isle, France) were housed in groups of 3 or 4 rats per cage on a 12 h/12 h day-night cycle (lights on at 8 am). Cage enrichment, in the form of a tunnel hanging from the top grid, was provided and food and water were available ad libitum. Experiments were carried out between 9 am and 5 pm. All experiments were approved by the KU Leuven animal ethics committee, and conducted in accordance with the Belgian Royal Decree of 29/05/2013 and European Directive 2010/63/EU.

2.4. Drug administration

Depending on the experiment (see Table 1), (-)-quinpirole HCl (QUIN; 0.05 mg/kg, 1 mg/kg or 5 mg/kg; Tocris), raclopride HCl (RACLO; 0.3 mg/kg; Santa Cruz Biotechnology), or saline was administered immediately after conditioning. Both drugs were dissolved in saline (0.9%, w/v) and injected intraperitoneally at a volume of 1 ml/kg. Drug doses were based on the study in mice by De Bundel et al. (2016). Similar doses have previously been used in rats (Boschen et al., 2011; Cobacho et al., 2014; Mueller et al., 2010; Woolley et al., 2003).

2.5. Differential cued fear conditioning

In brief, animals were first fear-conditioned in context A. This training session consisted of presentations of a danger-signaling tone (5 x CS+, always co-terminating with a foot shock (US)) and a safe tone (5 x CS-, never paired with the shock) in a semi-random order (see Table 1 and Fig. 1). Immediately after the end of the fear conditioning session, rats received an intraperitoneal injection of saline, quinpirole (0.05, 1 or 5 mg/kg) or raclopride (0.3 mg/kg) at a volume of 1 ml/kg. Twenty-four hours after training, rats underwent a drug-free test in a novel context B, during which the CS+ and CS- were repeatedly presented (4 times each) in a semi-random order. Depending on the experiment, additional drug-free test sessions were performed, in either context A or B.

Fig. 1. Experimental procedure.

Fig. 1

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Immediately after differential cued fear conditioning in context A (CTX-A), rats were systemically injected with saline, quinpirole, or raclopride (see Table 1 for details). Twenty-four hours later, during a drug-free test, fear responding to the CS+ and CS- was assessed in a novel context B (CTX-B, Test 1) using percentage of time spent freezing during the tones as an outcome measure, to evaluate the effects of the dopaminergic drug on subsequent fear generalization. Depending on the experiment, additional behavioral tests took place at least one day after Test 1.

2.5.1. Apparatus

Four chambers (Contextual NIR Video Fear Conditioning System for Rats, Med Associates Inc., St. Albans, VT, USA, 30 cm (L) x 25 cm (W) x 21 cm (H)) illuminated by infrared and white light (45 lux), and with built-in ventilation fans (±67 dB) were used. Fear conditioning took place in context A (CTX-A), consisting of a grid floor (19 rods of 4.8 mm diameter, 16 mm center to center) and a triangular-shaped black insert. Before and/or after each behavioral session, the context was cleaned and scented with diluted cleaning product (5.55 % in water). The first test session (Test 1) took place in context B, consisting of a white plastic floor, a white plastic curved back wall, illuminated with infrared light only, and cleaned and scented with a different cleaning product.

2.5.2. Training

Rats were placed in context A (CTX-A) and, after a baseline period of 120 s, two tones (5 and 10 kHz, counterbalanced) were presented five times each in semi-random order (80 dB, 30 s, ITI = 60 – 160 s, order of tone presentations = CS+, CS-, CS-, CS+, CS-, CS+, CS-, CS-, CS+, CS+). One tone (i.e., CS+) always co-terminated with a foot shock (.5 s,.4 or 1 mA, see Table 1 for shock intensities), while the other tone (i.e., CS-) was never paired with shock. Sixty seconds after the last tone, rats were returned to their home cage. Depending on the experiment, rats then immediately received an intraperitoneal injection of saline, quinpirole (0.05, 1 or 5 mg/kg) or raclopride (0.3 mg/kg) in 1 ml/kg (see Table 1).

2.5.3. Tests of fear responding to the CS+ and CS-

Twenty-four hours after training, rats underwent a drug-free test in a novel context (CTX-B). After a baseline period of 180 s, the CS+ and CS- tones were repeatedly presented in a semi-random and counterbalanced order (4 presentations of each tone, 80 dB, 30 s, ITI = 80 – 140 s, see Table 1 for the order of tone presentations). Thirty seconds after the last tone, rats were returned to their home cage.

Depending on the experiment, additional drug-free test sessions were performed (see Table 1). Two (Exp. 1) or 26 days (Exp. 2) after training, rats were re-exposed to the training context (CTX-A, Test 2), and, after a baseline period of 180 s, the CS+ and CS- tones were repeatedly presented in a semi-random and counterbalanced order (4 presentations of each tone, 80 dB, 30 s, ITI = 80 – 140 s). Thirty seconds after the last tone, rats were returned to their home cage. In Experiment 3, a second test took place in CTX-B at day 28, and a third test in CTX-A on Day 29. The order of the tones and ITIs differed between test sessions.

2.6. Statistical analyses

Percentage freezing during tones was scored by an experienced observer, blinded to treatment condition, tone frequency (5 or 10 kHz) and CS type (CS- or CS+). Average % freezing during CS+ and CS- was calculated for each rat and for each test session. According to a preregistered exclusion criterion, rats showing less than 10% freezing on average during CS+ presentations at Test 1 were excluded from the analysis, as these low freezing scores may indicate a lack of acquisition of the CS- US association. Analyses were performed with and without those rats. R was used for frequentist and Bayesian analyses. For Bayesian analyses, we used a default Cauchy prior on the standardized effect size with a scale of 0.707 in the BayesFactor package. Bayes factors indicate how likely the obtained data are under HA, relative to H0 (i.e., BF10).

Primary analyses for Experiments 1-5 (secondary analyses are described in the Supplement) included mixed ANOVAs with between-subjects factor Treatment (saline or drug) and within-subjects factor CS type (CS- or CS+) to investigate whether treatment affected fear responding to both tones during tests. Mixed ANOVAs were performed for each drug (versus saline) separately. The Bayes factor (BF) quantifying the evidence for including the Treatment x CS type interaction was calculated by dividing the BF for the model with interaction and main effects by the BF for the model with only main effects, (see doi 10.31234/osf.io/spreb; referred to as ‘BFinclusion across matched models’). One-sided paired t-tests assessed whether freezing during CS+ was higher compared to CS- for each treatment condition.

Primary statistical analyses for Experiment 5 were the same as for Exp. 1-4, but in contrast with the previous experiments, this study used a sequential design, for which the total sample size depended on the value of the Bayes factor for the CS type x Treatment interaction observed during interim assessments (see Table 2). The first assessment took place after testing a total of 40 rats (batch 1; n = 20 rats/treatment condition) and the second assessment after testing a total of 64 rats (batch 2; 24 additional rats; n = 12 rats/treatment condition). The maximum sample size (N = 88) was determined based on frequentist power calculations using the results obtained in Experiment 4 (CS Type x Treatment interaction; power = 92%, alpha =.05, partial eta squared =.13).

3. Results

All details regarding the behavioral protocol optimization studies (Experiments A-B-C) that preceded Experiments 1-5 can be found in the Supplement.

3.1. Experiment 1: No evidence for an effect of post-training injection of raclopride (0.3 mg/kg) or quinpirole (1 mg/kg) on subsequent fear generalization

Based on the results of De Bundel et al. (2016) in mice, we hypothesized that 0.3 mg/kg raclopride would increase and 1 mg/kg quinpirole would decrease fear generalization at test, respectively.

Primary analyses provided no evidence for Treatment (dopaminergic drug versus saline) x CS type (CS- versus CS+) interactions at Test 1 (RACLO: F(1, 22) =.04, p =.847, η2p <.01, BF =.38; QUIN1: F(1, 22) =.20, p =.662, η2p =.01, BF =.38). One-sided paired t-tests indicated higher freezing during CS+ than CS- presentations at test in saline control rats (t(11) = -2.42, p =.017, d = -.64, BF = 4.35), which, together with the considerable freezing levels during the CS-, suggested some differentiation, but also partial generalization at the group level. This intermediate level of generalization theoretically allowed for its augmentation or attenuation in the treatment groups. One-sided paired t-tests further indicated higher freezing during CS+ than CS- presentations at test in raclopride rats (t(11) = -2.6, p =.012, d = -.67, BF = 5.66), but not in quinpirole rats (t(11) = -1.38, p =.098, d = -.33, BF = 1.09), both in contrast with our hypotheses.

Although freezing during CS+ presentations at Test 1 was on average lower in quinpirole rats than in saline control rats (see Fig. 2a), two-sided t-tests (preregistered secondary analysis, see OSF) suggested no evidence for differences in freezing during CS+ presentations between treatment conditions (i.e., QUIN1 versus SAL or RACLO versus SAL). In addition, there was no evidence for differences in baseline freezing (i.e., before onset of the first tone, see OSF) or in the proportion of generalizers between treatment conditions.

Fig. 2. Experiment 1.

Fig. 2

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Average % freezing per treatment condition during Test 1 (Day 2; CTX-B; panel a) and Test 2 (Day 3; CTX-A; panel b), shown for the 180-s baseline period (pre), CS- and CS+. No significant group differences (i.e., no Treatment by CS type interactions) were found for Test 1 and Test 2. The results of one-sided paired t-tests (CS+ > CS-) are shown (*p <.05, **p <.01, NS = not significant). QUIN1 = quinpirole (1 mg/kg), RACLO = raclopride (0.3 mg/kg), SAL = saline.

During Test 2, which took place in the training context on Day 3 (Fig. 2b), there was again no evidence for Treatment x CS type interactions (RACLO: F(1, 22) =.67, p =.421, η2p =.03, BF =.45; QUIN1: F(1, 22) = 1.25, p =.276, η2p =.05, BF =.63) and one-sided paired t-tests suggested higher freezing during CS+ than CS- presentations at test in saline control rats (t(11) = -2.09, p =.03, d = -.60, BF = 2.75), raclopride rats (t(11) = -2.31, p =.021, d = -.29, BF = 3.75), and in quinpirole rats (t(11) = - 3.75, p =.002, d = -1.14, BF = 30.08). Secondary analyses suggested no evidence for differences in freezing during CS+ presentations between treatment conditions (i.e., QUIN1 versus SAL or RACLO versus SAL). In addition, there was no evidence for differences in baseline freezing (i.e., before onset of the first tone), average freezing during CS+ presentations, or the proportion of generalizers between treatment conditions.

3.2. Experiments 2 & 3: No evidence for an effect of post-training injection of quinpirole (0.05 or 5 mg/kg) on subsequent fear generalization

Locomotor activity studies in rats suggest that quinpirole differentially affects presynaptic versus postsynaptic D2 receptors depending on the applied dose (Eilam and Szechtman, 1989; Hartesveldt et al., 1992; Horvitz et al., 2001; Kropf and Kuschinsky, 1991). Experiments 2 and 3 aimed to assess how different doses of quinpirole influence fear generalization. We hypothesized that a low dose of quinpirole (0.05 mg/kg) might in fact increase fear generalization (due to the assumed activation of presynaptic autoreceptors), whereas a higher dose of 5 mg/kg would decrease fear generalization (due to the assumed activation of postsynaptic D2Rs). Experiments 2 and 3 adopted the exact same behavioral procedures and drug doses (apart from additional tests 2 and 3), and are therefore presented together.

In Experiment 2 (Fig. 3a-b), three rats (1 SAL and 2 QUIN5 rats) were excluded due to the preregistered exclusion criterion of showing less than 10% freezing on average during CS+ presentations at Test 1, so all preregistered analyses were performed with and without those three rats. Statistical results are shown here for the sample in which the exclusion criterion was applied, and results for both samples (i.e., with and without exclusions) are provided only when both analyses reached different overall conclusions. All results can be found at https://osf.io/kzcyt.

Fig. 3. Experiments 2 & 3.

Fig. 3

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Average % freezing per treatment condition during Test 1 (Day 2; CTX-B; panel a) and Test 2 (Day 27; CTX-A; panel b) of Experiment 2, and during Test 1 (Day 2; CTX-B; panel c), Test 2 (Day 28; CTX-B; panel d), and Test 3 (Day 29; CTX-A; panel e) of Experiment 3, shown for the 180-s baseline period (pre), CS- and CS+. No significant group differences (i.e., no Treatment by CS type interactions) were found for Test 1, 2 and 3. The results of one-sided paired t-tests (CS+ > CS-) are shown (*p <.05, **p <.01, NS = not significant). QUIN0.05 = quinpirole (0.05 mg/kg), QUIN5 = quinpirole (5 mg/kg), SAL = saline.

Primary analyses suggested no evidence for Treatment (quinpirole versus saline) x CS type (CS- versus CS+) interactions (QUIN (0.05 mg/kg): F(1, 21) = 2.01, p =.171, η2p =.09, BF =.89; QUIN (5 mg/kg): F(1, 19) =.53, p =.477, η2p =.03, BF =.48) at Test 1. Contrary to Experiment 1, in which the same behavioral procedures were used for training and testing as here, there was no statistically significant evidence for more freezing during CS+ compared to CS- presentations at test in saline control rats (t(10) = -.39, p =.353, d = -.12, BF =.40), suggesting more complete generalization in the control group of this experiment. A similar pattern of responding was observed in rats treated with the high dose of quinpirole (t(9) = -1.34, p =.106, d = -.51, BF = 1.1). In contrast, rats that received the low quinpirole dose did show significantly more freezing during the CS+ than during the CS- (t(11) = - 2.67, p =.011, d = -.59, BF = 6.25). Neither of the quinpirole doses thus induced the hypothesized effects on fear generalization. If anything, the low quinpirole dose (0.05 mg/kg) seemed to reduce, rather than augment, fear generalization, although this pattern did not reach statistical significance.

Average freezing during CS+ presentations at Test 1 was numerically higher in the ‘low quinpirole’ condition than in saline control rats (see Fig. 3a), but this between-group difference did not receive statistical support when excluding the saline rat showing on average less than 10% freezing during CS+ presentations (t(21) = 1.79, p =.088, d =.75, BF = 1.16). When including all rats, the effect was statistically significant, although the obtained evidence was indifferent according to the Bayesian analysis (t(22) = 2.1, p =.047, d =.86, BF = 1.72). Furthermore, no between-group differences were found in baseline freezing (i.e., before onset of the first tone) or the proportion of generalizers.

During Test 2, which took place in the training context on Day 27 (Fig. 3b), there was again no evidence for Treatment x CS type interactions (QUIN (0.05 mg/kg): F(1, 21) =.06, p =.801, η2p <.01, BF =.37; QUIN (5 mg/kg): F(1, 19) <.01, p =.945, η2p <.01, BF =.38). Furthermore, both QUIN0.05 and saline rats now showed significantly higher freezing during CS+ than CS- (t(10) = -2.96, p =.007, d = -.77, BF = 8.98; t(11) = -3.85, p =.001, d = -1.20, BF = 34.85, respectively), whereas QUIN5 rats did not show statistically significant differential responding (t(9) = -1.61, p =.071, d = -.63, BF = 1.51). In addition, secondary analyses suggested no evidence for differences in baseline freezing (i.e., before onset of the first tone), average freezing during CS+ presentations, or the proportion of generalizers between treatment conditions.

In Experiment 3 (Fig. 3c-e), eight rats (2 SAL, 2 QUIN0.05, and 4 QUIN5 rats) were excluded due to the preregistered exclusion criterion of showing less than 10% freezing on average during CS+ presentations at Test 1, so all preregistered analyses were performed with (N = 36) and without (N = 28) those eight rats.

Primary analyses suggested no evidence for Treatment (dopaminergic drug versus saline) x CS type (CS- versus CS+) interactions (QUIN (0.05 mg/kg): F(1, 18) =.55, p =.468, η2p =.03, BF =.47; QUIN (5 mg/kg): F(1, 16) =.88, p =.362, η2p =.05, BF =.54) at Test 1 (Fig. 3c). One-sided paired t-tests suggested higher freezing during CS+ than CS- presentations at test in all treatment conditions (saline control rats: (t(9) = -3.64, p =.003, d = -.42, BF = 20.35; QUIN (0.05 mg/kg): t(9) = -2.82, p =.01, d = -.76, BF = 7.03; QUIN (5 mg/kg): t(7) = -3.52, p =.005, d = -.98, BF = 13.27). The difference in fear responding between CS+ and CS- was smaller (and non-significant) when including all QUIN5 rats (t(22) =.08, p =.933, d =.03, BF =.37), probably due to the fact that two rats showed no freezing during any of the tones, and thus, no differential responding.

During Test 2 (Fig. 3d), which took place in the testing context (CTX-B) on Day 28, there was again no evidence for Treatment x CS type interactions (QUIN0.05: F(1, 18) =.57, p =.458, η2p =.03, BF =.47; QUIN5: F(1, 16) =.03, p =.861, η2p <.01, BF =.39). Saline control rats (t(9) = -1.25, p =.121, d = -.24, BF =.99) and QUIN5 rats (t(7) = -1.37, p =.107, d = -.77, BF = 1.18) did no longer show significantly more freezing to the CS+ than to the CS-, whereas QUIN0.05 rats still did (t(9) = -2.76, p =.011, d = -.78, BF = 6.47). However, when including all 36 rats, all groups showed significantly more freezing to the CS+ than to the CS- (SAL: t(11) = -1.86, p =.045, d = -.33, BF = 2.01; QUIN0.05: t(11) = - 2.48, p =.015, d = -.61, BF = 4.72; QUIN5: t(11) = -2.11, p =.029, d = -.65, BF = 2.83).

During Test 3 (Fig. 3e), which took place in the training context (CTX-A) on Day 29, there was no evidence for Treatment x CS type interactions (QUIN0.05: F(1, 18) <.01, p =.982, η2p <.01, BF =.41; QUIN5: F(1, 16) =.74, p =.402, η2p =.04, BF =.58) and there was no statistical evidence for differences in responding to the CS+ and CS- in any of the treatment conditions (SAL: t(9) = -1.45, p =.09, d = -.5, BF = 1.25; QUIN0.05: t(9) = -1.48, p =.086, d = -.53, BF = 1.29; QUIN5: t(7) =.08, p =.531, d =.04, BF =.32).

Secondary analyses provided no evidence for between-group differences in average freezing during CS+ presentations, baseline freezing, (i.e., before onset of the first tone), or the proportion of generalizers during any of the test sessions.

3.3. Experiment 4: No evidence for an effect of post-training injection of quinpirole (0.05 or 1 mg/kg) on fear generalization when using higher shock intensity during training

Experiment 4 aimed to investigate whether quinpirole injection can prevent fear generalization at a drug-free test when using a higher shock intensity during training. We expected that this higher shock intensity would result in more fear generalization at test in saline control rats than in our prior experiments (based on the results of Experiment C, see Supplement). Such a saline group would also be more similar to the saline condition in the mouse study by De Bundel and colleagues (2016). Note that, although at the start of our study, we hypothesized that the low dose of quinpirole (i.e., 0.05 mg/kg) would increase fear generalization, the results of Exp. 2 suggested that, if anything, this dose may prevent fear generalization. Therefore, the low dose was also used in this study, to examine if it would prevent fear generalization, similar to the hypothesis for the 1 mg/kg dose.

Despite the use of stronger shocks, saline control rats showed significantly more freezing to the CS+ than to the CS- at test (t(15) = -1.8, p =.046, d = -.35, BF = 1.78), and thus still incomplete fear generalization at the group level. Also in quinpirole rats, the difference in freezing to the CS+ and CS- was significant, and numerically even higher, especially in rats that received the dose of 1 mg/kg (QUIN0.05: t(15) = -2.34, p =.017, d = -.44, BF = 4.01; QUIN1: t(15) = -5.04, p <.001, d = -.86, BF = 410.27) (Fig. 4a). If significant, such increased differentiation between CS+ and CS- would be in line with our hypothesis that quinpirole should attenuate fear generalization. There was, however, no reliable evidence for Treatment (dopaminergic drug versus saline) x CS type (CS- versus CS+) interactions (QUIN (0.05 mg/kg): F(1, 30) =.25, p =.622, η2p =.01, BF =.36; QUIN (1 mg/kg): F(1, 30) = 3.28, p =.08, η2p =.1, BF = 1.08).

Fig. 4. Experiments 4 & 5.

Fig. 4

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Average % freezing per treatment condition during testing (Day 2; CTX-B) in Experiment 4 (panel a) and Experiment 5 (panel b), shown for the 180-s baseline period (pre), CS- and CS+. No significant group differences (i.e., no Treatment by CS type interactions) were found during Test. The results of one-sided paired t-tests (CS+ > CS-) are shown (*p <.05, ***p <.001). QUIN0.05 = quinpirole (0.05 mg/kg), QUIN1 = quinpirole (1 mg/kg), SAL = saline.

Secondary analyses provided no evidence for between-group differences in average freezing during CS+ presentations, baseline freezing, (i.e., before onset of the first tone), or the proportion of generalizers.

3.4. Experiment 5: No evidence for an effect of post-training injection of quinpirole (1 mg/kg) on fear generalization when using higher shock intensity during training and large sample size

In Experiment 4 we observed numerically weaker generalization between the CS+ and CS- in rats that received 1 mg/kg quinpirole than in saline controls. Therefore, we aimed to investigate this effect further by taking into account the effect size observed in Exp. 4 and using a sample size that is required to detect such an effect with a power of more than 90%. Experiment 5 followed the procedures from Experiment 4, but only included two of the groups from Experiment 4 (i.e., quinpirole 1 mg/kg versus saline). The experiment had a sequential design with a predetermined maximum sample size, and was performed in three separate batches. Given that we did not observe the predefined Bayes factor for the CS type x Treatment interaction after testing batch 1 or 2, the maximum sample size of 88 rats was tested.

One saline rat was excluded from all analyses because it showed no freezing at all during testing (i.e., 0% freezing; exclusion based upon preregistered criterion). In addition, the excluded rat did not show any freezing during training either, and a lack of responding at the time the shocks should have been given, so we decided not to perform analyses with this rat included. Note that we hereby deviate from the preregistration because we planned to perform the analyses with all subjects included, apart from applying the preregistered exclusion criterion. A total of 87 rats was thus included in the analyses described below.

There was no evidence for a CS type x Treatment interaction (F(1, 85) =.17, p =.68, η2p <.01, BF =.23), and the obtained Bayes factor suggested substantial evidence for the absence of an interaction (Wetzels et al., 2011) (Fig. 4b). One-sided paired t-tests suggested significantly more freezing to the CS+ than the CS- in saline control rats (t(42) = -4.93, p <.001, d = -.62, BF = 3032.43) and quinpirole rats (t(43) = -4.31, p <.001, d = -0.56, BF = 502.73).

Secondary analyses suggested no difference in the proportion of generalizers between saline and quinpirole rats (X2(1) <.01, p = 1, BF =.27). In addition, the generalization index was not significantly lower in quinpirole rats than in saline rats (t(85) =.76, p =.777, d =.16, BF =.14).

3.5. Experiment 6: Quinpirole (0.05 mg/kg, 1 mg/kg, 5 mg/kg) and raclopride (0.3 mg/kg) affect locomotor activity

As mentioned before, we carried out an additional study to assess the acute effects of quinpirole and raclopride on locomotor activity in a novel arena (38 cm x 38 cm). Drugs were injected 5 min before the start of the locomotor test. All details regarding the methods and results can be found in the Supplement. In brief, we observed locomotor effects that were largely in line with prior research. Importantly, as described in the literature (Horvitz et al., 2001), we found that 1 mg/kg quinpirole first suppressed locomotor activity, followed by a statistically significant increase in locomotor activity compared to saline (Fig. 5). This indicates, that in a different task than the fear conditioning procedure, the drug did produce the expected acute behavioral effect at the applied dose in male Wistar rats.

Fig. 5. Experiment 6.

Fig. 5

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Locomotor activity (distance travelled in cm), starting 5 min after injection with saline, quinpirole (0.05 mg/kg, 1 mg/kg, 5 mg/kg) or raclopride (0.3 mg/kg). Panel a shows activity (means and SEMs) throughout the 1-hour test session. Panel b shows locomotor activity during the first 5 minutes of the session (preregistered one-sided t-tests indicating significant suppression of locomotor activity in comparison with the saline group, ***p <.001). Panel c shows locomotor activity during the last 5 minutes of the session (preregistered one-sided t-test indicating significantly increased locomotor activity as compared with the saline group, *p <.05). QUIN0.05 = quinpirole (0.05 mg/kg), QUIN1 = quinpirole (1 mg/kg), QUIN5 = quinpirole (5 mg/kg), RACLO = raclopride (0.3 mg/kg), SAL = saline.

4. Discussion

In a series of five experiments, the dopamine D2 receptor (D2R) agonist quinpirole or D2R antagonist raclopride was injected systemically after differential cued fear conditioning in rats. One day after treatment, fear generalization was assessed by comparing average % freezing during CS+ versus CS- presentations in a new context. Our studies aimed to evaluate in rats the role of dopaminergic D2R signaling in fear memory processing as established in mice, emulating the protocol of a previously published study by De Bundel and colleagues (2016). They showed that post-training systemic injection in mice of quinpirole (1 mg/kg) or raclopride (0.3 mg/kg) differentially affected fear responding to a learned danger cue (CS+) versus safety cue (CS-). Based on those published results, we expected quinpirole (1 or 5 mg/kg) to prevent fear generalization, and raclopride (0.3 mg/kg) to augment fear generalization. In addition, we initially hypothesized that a low dose of quinpirole (0.05 mg/kg) would also augment fear generalization, due to the assumed activation of presynaptic autoreceptors. We found no evidence in rats for an effect of systemically administered quinpirole or raclopride on fear generalization at test. In addition, there were no effects of treatment on freezing levels during CS+ presentations per se or during the baseline period at test (i.e., before tone onset) in any of the 5 experiments. We did replicate previously described acute behavioral effects of quinpirole (0.05, 1 and 5 mg/kg) and raclopride (0.3 mg/kg) during a locomotor activity test, indicating that the applied drug doses and route of administration were capable of inducing previously reported behavioral effects.

In the light of the inconsistent results between our studies (in rats) and the published studies by De Bundel et al. (2016) (in mice), a number of differences between these two studies should be acknowledged. First and foremost is of course the difference in species, given the reported species-dependent variations in ventral tegmental area dopamine D2R signaling (Courtney et al., 2012). The fact that DA neurons in mice and rats show different sensitivity to D2-mediated inhibition could have important implications for presynaptic or postsynaptic effects of D2R modulation. Similarly, differences in DA agonist-induced locomotor effects, prepulse inhibition, or yawning between rats and mice have been described (Halberda et al., 1997; Li et al., 2010; Ralph and Caine, 2005). It should also be noted that, although the doses applied in our study are identical to those used for mice by De Bundel et al. (2016) and have previously been used in rats, we cannot exclude that the pharmacokinetics and pharmacodynamics differ between species. Another relevant question is whether mice and rats express fear in the same manner (e.g., comparable levels of freezing versus escape behavior) during presentations of tones with different frequencies. If existent, between-species differences in prototypical fear behavior may obviously complicate the comparison of studies in rats versus mice when freezing responses to the CS+ versus CS- are used as a measure of generalization.

Furthermore, and as expected, pilot studies in our lab indicated the need for different behavioral parameters than those used in the mouse studies (De Bundel et al., 2016) in order to allow for the study of fear generalization in rats (e.g., tones of 5 and 10 kHz rather than 2.5 and 7.5 kHz, shocks of.5 s rather than 2 s, and intermixed rather than blocked testing). In addition, we chose to leave out the habituation session prior to conditioning, and used a semi-random rather than an alternating order of CS+ and CS- presentations during learning. Finally, our saline control animals showed, on average, a different behavioral pattern than those in the De Bundel study. The control rats in most of our dopamine studies (except for Experiment 2) showed significantly higher freezing during CS+ presentations than during CS-, whereas the control mice in the prior quinpirole study did not freeze more to the CS+ than to the CS- (see Fig. 1C of De Bundel et al. (2016)). Nevertheless, it is clear that our control rats showed fear generalization, as freezing to the CS- was considerable, thus fulfilling the prerequisite to be able to observe any effect on fear generalization in the quinpirole groups. The observed variability in freezing behavior of the control animals between some of our experiments could be perceived as a limitation, but is probably a logical consequence of our attempt to tread a fine line, i.e., to create a learning experience that still contained a degree of ambiguity (with the control animals of Experiment 2 showing statistically insignificant CS+ versus CS- differentiation, and thus flipping to the other side than in Experiments A, 1 and 3, where the difference was significant). As mentioned before, we did not want to overtrain the animals in their discrimination between the CS+ and CS- (which could, for example, have been achieved with multiple training days (Day et al., 2016)), nor did we want them to have inadequate learning opportunities to differentiate between the CS+ and CS- (in such scenario, full generalization between the CS+ and CS- might just reflect a perceptual discrimination deficit). Overall, the observed behavioral variability between experiments is most likely not a major issue, given that each of our experiments had its own appropriate saline control group.

A potential limitation of our study is that we focused on systemic drug injection and did not directly target a particular brain region via, for example, local drug infusion. Given the apparent lack of generalizability to rats of the results of De Bundel et al. (2016) of systemic application of dopaminergic drugs, a follow-up study in rats may indeed want to use intracerebral drug administration (as was done in mice in Fig. 2I and 3I of De Bundel et al. (2016)). Such a study will allow for even stronger conclusions about the replicability (and generalizability) of the effects described in mice specifically, and about the role of post-training D2R signaling in subsequent fear generalization in rats more generally.

In summary, contrary to our hypothesis, we did not find systemic raclopride (0.3 mg/kg) to augment fear generalization, although it should be noted that this conclusion merely relies on a single study with one dose and with a sample size of 12 rats per treatment condition, yielding only anecdotal evidence for the absence of a CS type x Treatment interaction effect. In contrast, we performed five studies with quinpirole, in which we used three different doses of the drug (0.05, 1, or 5 mg/kg), two different shock intensities (.4 or 1 mA), and including a final study with a large sample size yielding a power of over 90% to detect an effect if any. Moreover, Experiment 3 (N = 36) was a replication of Experiment 2 (N = 36) (except for additional tests), and Experiment 5 (N = 88) was a replication of Experiment 4 (N = 48) (except for the omission of the 0.05-mg/kg quinpirole group in Exp. 5). The final experiment provided substantial evidence (Bayesian analyses) for the absence of an effect of quinpirole (1 mg/kg) on fear responding to the CS+ versus CS-. In other words, we have gathered evidence against a preventative (or augmentative) effect of dopamine D2 receptor agonism with quinpirole on fear generalization, under the applied conditions in rats.

Supplementary Material

Supplementary Materials

Funding

Funding for this study was provided by the European Research Council (ERC Consolidator Grant to T. Beckers, grant number 648176), the Research Foundation – Flanders, Belgium (FWO Doctoral Fellowship to N. Schroyens, grant number 1114018N) and KU Leuven Research Grant C16/19/002 (to T. Beckers and L. Luyten). The funders had no further role in study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the paper for publication.

Footnotes

Conflicts of interest: None.

Contributors

Conceptualization: N.S., T.B. and L.L.; Data curation: N.S.; Formal analysis: N.S.; Funding acquisition: N.S., T.B. and L.L.; Investigation: N.S., L.V., B.Ö. and V.A.O.S.; Methodology: N.S., J.Z., D.D., T.B. and L.L.; Project administration: T.B. and L.L.; Resources: T.B.; Supervision: T.B. and L.L.; Validation: N.S.; Visualization: N.S. and L.L.; Writing – original draft: N.S. and L.L.; Writing - review & editing: N.S., L.V., B.Ö., V.A.O.S., J.Z., D.D., T.B. and L.L. All authors contributed to and have approved the final manuscript.

Conflict of Interest

All authors declare that they have no conflicts of interest.

2.2. Availability of data and materials

All datasets generated and analyzed during the current study, the analysis scripts, and results of all preregistered statistical analyses are also available on OSF (https://osf.io/kzcyt). This study is reported in accordance with ARRIVE guidelines (https://arriveguidelines.org).

References

  1. Bissière S, Humeau Y, Lüthi A. Dopamine gates LTP induction in lateral amygdala by suppressing feedforward inhibition. Nature Neuroscience. 2003;6:587–592. doi: 10.1038/nn1058. [DOI] [PubMed] [Google Scholar]
  2. Boschen SL, Wietzikoski EC, Winn P, Cunha CD. The role of nucleus accumbens and dorsolateral striatal D2 receptors in active avoidance conditioning. Neurobiology of Learning and Memory. 2011;96:254–262. doi: 10.1016/j.nlm.2011.05.002. [DOI] [PubMed] [Google Scholar]
  3. Castellano C, Cestari V, Cabib S, Puglisi-Allegra S. Post-training dopamine receptor agonists and antagonists affect memory storage in mice irrespective of their selectivity for D1 or D2 receptors. Behavioral and Neural Biology. 1991;56:283–291. doi: 10.1016/0163-1047(91)90439-W. [DOI] [PubMed] [Google Scholar]
  4. Cobacho N, de la Calle JL, Paíno CL. Dopaminergic modulation of neuropathic pain: Analgesia in rats by a D2-type receptor agonist. Brain Research Bulletin. 2014;106:62–71. doi: 10.1016/j.brainresbull.2014.06.003. [DOI] [PubMed] [Google Scholar]
  5. Colucci P, Mancini GF, Santori A, Zwergel C, Mai A, Trezza V, Roozendaal B, Campolongo P. Amphetamine and the Smart Drug 3,4-Methylenedioxypyrovalerone (MDPV) Induce Generalization of Fear Memory in Rats. Frontiers in Molecular Neuroscience. 2019;12 doi: 10.3389/fnmol.2019.00292. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Courtney NA, Mamaligas AA, Ford CP. Species differences in somatodendritic dopamine transmission determine D2-autoreceptor-mediated inhibition of ventral tegmental area neuron firing. Journal of Neuroscience. 2012;32:13520–13528. doi: 10.1523/JNEUROSCI.2745-12.2012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Day HLL, Reed MM, Stevenson CW. Sex differences in discriminating between cues predicting threat and safety. Neurobiol Learn Mem. 2016;133:196–203. doi: 10.1016/j.nlm.2016.07.014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. De Bundel D, Zussy C, Espallergues J, Gerfen CR, Girault J-A, Valjent E. Dopamine D2 receptors gate generalization of conditioned threat responses through mTORC1 signaling in the extended amygdala. Molecular Psychiatry. 2016;21:1545–1553. doi: 10.1038/mp.2015.210. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Eilam D, Szechtman H. Biphasic effect of D-2 agonist quinpirole on locomotion and movements. European Journal of Pharmacology. 1989;161:151–157. doi: 10.1016/0014-2999(89)90837-6. [DOI] [PubMed] [Google Scholar]
  10. Ellenbroek B, Youn J. Rodent models in neuroscience research: is it a rat race? Disease Models & Mechanisms. 2016;9:1079–1087. doi: 10.1242/dmm.026120. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fadok, Fadok Dickerson TMK, Palmiter RD. Dopamine is necessary for cue-dependent fear conditioning. Journal of Neuroscience. 2009;29:11089–11097. doi: 10.1523/JNEUROSCI.1616-09.2009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Frey U, Matthies Henry, Reymann KG, Matthies Hansjürgen. The effect of dopaminergic D1 receptor blockade during tetanization on the expression of long-term potentiation in the rat CA1 region in vitro. Neuroscience Letters. 1991;129:111–114. doi: 10.1016/0304-3940(91)90732-9. [DOI] [PubMed] [Google Scholar]
  13. Frick A, Björkstrand J, Lubberink M, Eriksson A, Fredrikson M, Åhs F. Dopamine and fear memory formation in the human amygdala. Mol Psychiatry. 2021 doi: 10.1038/s41380-021-01400-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Greba Q, Gifkins A, Kokkinidis L. Inhibition of amygdaloid dopamine D2 receptors impairs emotional learning measured with fear-potentiated startle. Brain Research. 2001;899:218–226. doi: 10.1016/S0006-8993(01)02243-0. [DOI] [PubMed] [Google Scholar]
  15. Halberda JP, Middaugh LD, Gard BE, Jackson BP. DAD1- and DAD2-like agonist effects on motor activity of C57 mice: Differences compared to rats. Synapse. 1997;26:81–92. doi: 10.1002/(SICI)1098-2396(199705)26:1&#x0003c;81&#x02237;AID-SYN9&#x0003e;3.0.CO;2-A. [DOI] [PubMed] [Google Scholar]
  16. Hartesveldt CV, Cottrell GA, Potter T, Meyer ME. Effects of intracerebral quinpirole on locomotion in rats. European Journal of Pharmacology. 1992;214:27–32. doi: 10.1016/0014-2999(92)90091-H. [DOI] [PubMed] [Google Scholar]
  17. Hermans D, Baeyens F, Vervliet B. Generalization of acquired emotional responses. Handbook of Cognition and Emotion. 2013:117–134. [Google Scholar]
  18. Horvitz JC, Williams G, Joy R. Time-dependent actions of D2 family agonist quinpirole on spontaneous behavior in the rat: Dissociation between sniffing and locomotion. Psychopharmacology. 2001;154:350–355. doi: 10.1007/s002130000677. [DOI] [PubMed] [Google Scholar]
  19. Iemolo A, Risi M, De Leonibus E. Role of dopamine in memory consolidation. Memory Consolidation. 2015:161–197. [Google Scholar]
  20. Jo YS, Heymann G, Zweifel LS. Dopamine Neurons Reflect the Uncertainty in Fear Generalization. Neuron. 2018;100:916–925.:e3. doi: 10.1016/j.neuron.2018.09.028. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Jones GL, Soden ME, Knakal CR, Lee H, Chung AS, Merriam EB, Zweifel LS. A genetic link between discriminative fear coding by the lateral amygdala, dopamine, and fear generalization. eLife. 2015;4 doi: 10.7554/eLife.08969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kropf W, Kuschinsky K. Electroencephalographic correlates of the sedative effects of dopamine agonists presumably acting on autoreceptors. Neuropharmacology. 1991;30:953–960. doi: 10.1016/0028-3908(91)90108-N. [DOI] [PubMed] [Google Scholar]
  23. LaLumiere RT, Nguyen LT, McGaugh JL. Post-training intrabasolateral amygdala infusions of dopamine modulate consolidation of inhibitory avoidance memory: involvement of noradrenergic and cholinergic systems. European Journal of Neuroscience. 2004;20:2804–2810. doi: 10.1111/j.1460-9568.2004.03744.x. [DOI] [PubMed] [Google Scholar]
  24. Lee JC, Wang LP, Tsien JZ. Dopamine rebound-excitation theory: Putting brakes on PTSD. Front Psychiatry. 2016;7 doi: 10.3389/fpsyt.2016.00163. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Lemon N, Manahan-Vaughan D. Dopamine D1/D5 receptors gate the acquisition of novel Information through hippocampal long-term potentiation and long-term depression. Journal of Neuroscience. 2006;26:7723–7729. doi: 10.1523/JNEUROSCI.1454-06.2006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Li S-M, Collins GT, Paul NM, Grundt P, Newman AH, Xu M, Grandy DK, Woods JH, Katz JL. Yawning and locomotor behavior induced by dopamine receptor agonists in mice and rats. Behavioural Pharmacology. 2010;21:171–181. doi: 10.1097/FBP.0b013e32833a5c68. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Lisman JE, Grace AA. The hippocampal-VTA loop: Controlling the entry of Information into long-term memory. Neuron. 2005;46:703–713. doi: 10.1016/j.neuron.2005.05.002. [DOI] [PubMed] [Google Scholar]
  28. Manago F, Castellano C, Oliverio A, Mele A, De Leonibus E. Role of dopamine receptors subtypes, D1-like and D2-like, within the nucleus accumbens subregions, core and shell, on memory consolidation in the one-trial inhibitory avoidance task. Learning & Memory. 2008;16:46–52. doi: 10.1101/lm.1177509. [DOI] [PubMed] [Google Scholar]
  29. Moaddab M, McDannald MA. Retrorubral field is a hub for diverse threat and aversive outcome signals. Curr Biol. 2021;31:2099–2110.:e5. doi: 10.1016/j.cub.2021.02.055. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Mueller D, Bravo-Rivera C, Quirk GJ. Infralimbic D2 receptors are necessary for fear extinction and extinction-related tone responses. Biological Psychiatry. 2010;68:1055–1060. doi: 10.1016/j.biopsych.2010.08.014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Ng KH, Pollock MW, Urbanczyk PJ, Sangha S. Altering D1 receptor activity in the basolateral amygdala impairs fear suppression during a safety cue. Neurobiology of Learning and Memory. 2018;147:26–34. doi: 10.1016/j.nlm.2017.11.011. [DOI] [PubMed] [Google Scholar]
  32. Ralph RJ, Caine SB. Dopamine D1 and D2 agonist effects on prepulse inhibition and locomotion: Comparison of Sprague-Dawley rats to Swiss-Webster, 129X1/SvJ, C57BL/6J, and DBA/2J mice. J Pharmacol Exp Ther. 2005;312:733–741. doi: 10.1124/jpet.104.074468. [DOI] [PubMed] [Google Scholar]
  33. Robertson EM. Memory instability as a gateway to generalization. PLOS Biology. 2018;16:e2004633. doi: 10.1371/journal.pbio.2004633. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Rosenkranz JA, Grace AA. Dopamine-mediated modulation of odour-evoked amygdala potentials during pavlovian conditioning. Nature. 2002;417:282–287. doi: 10.1038/417282a. [DOI] [PubMed] [Google Scholar]
  35. Rossato JI, Bevilaqua LRM, Izquierdo I, Medina JH, Cammarota M. Dopamine Controls Persistence of Long-Term Memory Storage. Science. 2009;325:1017–1020. doi: 10.1126/science.1172545. [DOI] [PubMed] [Google Scholar]
  36. Sarinana J, Kitamura T, Kunzler P, Sultzman L, Tonegawa S. Differential roles of the dopamine 1-class receptors, D1R and D5R, in hippocampal dependent memory. Proceedings of the National Academy of Sciences. 2014;111:8245–8250. doi: 10.1073/pnas.1407395111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Wetzels R, Matzke D, Lee MD, Rouder JN, Iverson GJ, Wagenmakers E-J. Statistical evidence in experimental psychology: An empirical comparison using 855 t Tests. Perspect Psychol Sci. 2011;6:291–298. doi: 10.1177/1745691611406923. [DOI] [PubMed] [Google Scholar]
  38. Woolley ML, Marsden CA, Sleight AJ, Fone KCF. Reversal of a cholinergic-induced deficit in a rodent model of recognition memory by the selective 5-HT6 receptor antagonist, Ro 04-6790. Psychopharmacology (Berl) 2003;170:358–367. doi: 10.1007/s00213-003-1552-5. [DOI] [PubMed] [Google Scholar]
  39. Yau JO-Y, McNally GP. The activity of ventral tegmental area dopamine neurons during shock omission predicts safety learning. Behav Neurosci. 2022;136:276–284. doi: 10.1037/bne0000506. [DOI] [PubMed] [Google Scholar]
  40. Zweifel LS, Fadok JP, Argilli E, Garelick MG, Jones GL, Dickerson TMK, Allen JM, Mizumori SJY, Bonci A, Palmiter RD. Activation of dopamine neurons is critical for aversive conditioning and prevention of generalized anxiety. Nature Neuroscience. 2011;14:620–626. doi: 10.1038/nn.2808. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Materials
EMS197949-supplement-Supplementary_Materials.pdf (594.7KB, pdf)

Data Availability Statement

All datasets generated and analyzed during the current study, the analysis scripts, and results of all preregistered statistical analyses are also available on OSF (https://osf.io/kzcyt). This study is reported in accordance with ARRIVE guidelines (https://arriveguidelines.org).

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