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Published in final edited form as: Neurobiol Learn Mem. 2023 Dec 20;208:107881. doi: 10.1016/j.nlm.2023.107881

Conditioned Inhibition of Fear and Reward in Male and Female Rats

Jamie N Krueger 1, Nupur N Patel 1, Kevin Shim 1, Ka Ng 1, Susan Sangha 1,2,*
PMCID: PMC10922191  NIHMSID: NIHMS1956110  PMID: 38135111

Abstract

Stimuli in our environment are not always associated with an outcome. Some of these stimuli, depending on how they are presented, may gain inhibitory value or simply be ignored. If experienced in the presence of other cues predictive of appetitive or aversive outcomes, they typically gain inhibitory value and become predictive cues indicating the absence of appetitive or aversive outcomes. In this case, these cues are referred to as conditioned inhibitors. Here, male and female Long Evans rats underwent cue discrimination training where a reward cue was paired with sucrose, a fear cue with footshock, and an inhibitor cue resulted in neither sucrose or footshock. During a subsequent summation test for conditioned inhibition of fear and reward, the inhibitor cue was presented concurrently with the reward and fear cues without any outcome, intermixed with trials of reinforced reward and fear trials. Males showed significant conditioned inhibition of freezing, while females did not, which was not dependent on estrous. Both males and females showed significant conditioned inhibition of reward. During a retardation of fear acquisition test, the inhibitor was paired with footshock and both males and females showed delayed acquisition of fear. During a retardation of reward acquisition test, the inhibitor was paired with sucrose, and females showed delayed acquisition of reward, while males did not. In summary, males and females showed significant reward-fear-inhibitor cue discrimination, conditioned inhibition of reward, and retardation of fear acquisition. The main sex difference, which was not estrous-dependent, was the lack of conditioned inhibition of freezing in females. These data imply that while the inhibitor cue gained some inhibitory value in the females, the strength of this inhibitory value may not have been great enough to effectively downregulate freezing elicited by the fear cue.

Keywords: Conditioned inhibition, safety conditioning, sex differences, fear, reward

Introduction

A range of psychiatric disorders have been linked to disrupted conditional discrimination and inhibition of fear (De Kleine et al., 2023; Jovanovic et al., 2009, 2012; Rabinak et al., 2017; Thurston & Cassaday, 2022). Stimuli in our environment that are not associated with any salient outcome can develop inhibitory value, especially if in the temporal vicinity of stimuli that are associated with salient outcomes. A stimulus that has gained inhibitory value as a result of conditioning has the power to counteract expectations and behaviors elicited by stimuli that have gained excitatory value. At its core, inhibitory cues are learned when the CS is associated with no US. However, simply presenting a CS without a US does not guarantee that it becomes a conditioned inhibitor. For example, pre-exposure to the CS without a US before conditioning begins, i.e. latent inhibition, may result in delayed learning when it is later paired with a US, but typically is not successful in counteracting behavior in response to an excitatory CS. To be a true conditioned inhibitor, the CS needs to pass both a ‘summation’ test and ‘retardation of acquisition’ test (Rescorla, 1969a). An inhibitor presented in compound with an excitor should ‘summate’ to reduce behavioral responding compared to when the excitor is presented alone. Further, an inhibitor that is later paired with a US, should show ‘retarded acquisition’ or a slower development of the conditioned behavior that an initially neutral CS paired with a US would produce.

If a CS resulting in no US does not gain inhibitory properties, but instead is learned to be ignored, it would pass the retardation test but not the summation test (Rescorla, 1969a), similar to latent inhibition. Conversely, if there is increased attention paid to the CS without it gaining inhibitory properties, one may expect it to pass the summation test but not the retardation test (Rescorla, 1969a). Combining the two tests makes for a powerful argument that a CS is indeed a conditioned inhibitor. CSs trained as inhibitors can be applied against excitors that were conditioned with aversive or appetitive outcomes. That is, an aversive excitor paired with a conditioned inhibitor should result in less fear expression (Rescorla, 1969c, 1971), while an appetitive excitor paired with a conditioned inhibitor should result in less reward-seeking behaviors (Tobler et al., 2003). This has been shown for aversive and appetitive paradigms and is reviewed in (Cassaday et al., 2023). However, to our knowledge, no one has integrated both aversive and appetitive excitors with a conditioned inhibitor in the same paradigm.

Post-traumatic Stress Disorder (PTSD) represents a condition where conditional discrimination and inhibition of fear have been shown to be particularly disrupted (De Kleine et al., 2023; Jovanovic & Norrholm, 2011; Jovanovic et al., 2009, 2012; Kribakaran et al., 2022; Rabinak et al., 2017). However, even in healthy adults, we have shown the perception of the inhibitor can impact physiological responses to the inhibitor when presented in compound with either an aversive or appetitive excitor (Fitzgerald et al., 2023). For example, the more favorable the inhibitor was rated, the greater the reduction in skin conductance response to the inhibitor when in compound with the fear cue. Since others have shown that individuals with anxiety-related disorders report increased expectancy of threat following a safety cue (De Kleine et al., 2023; Rabinak et al., 2017), a task combining conditioned inhibition of fear and reward has potentially high translational significance.

Here, male and female Long Evans rats underwent cue discrimination training where a reward cue was paired with sucrose, a fear cue with footshock, and an inhibitor cue resulted in neither sucrose or footshock. The inhibitor cue was then tested for conditioned inhibition in a subsequent summation test where it was concurrently presented with the fear and reward cues. A subset of rats then underwent a test for retardation of fear acquisition where the inhibitor was paired with shock, while another underwent a test for retardation of reward acquisition where the inhibitor was paired with sucrose.

Materials and Methods

Subjects

24 male and 24 age-matched female Long Evans rats (Blue Spruce; Envigo, Indianapolis) weighing 250–275g or 175–199g, respectively, upon arrival were single-housed under a 12hr light/dark cycle (lights on 09:00), acclimated to housing conditions for 1 week, and then handled for 1 week before commencing experiments. All procedures were performed during the light cycle and approved by the Purdue Animal Care and Use Committee. Rats had ad libitum access to food and water up until the first training session, at which point they received 20–22g (males) or 18–20g (females) of food per day after their daily training session for the remainder of the experiment. Most rats still had food at the time of the next feeding.

Apparatus

The training chambers were 12 Med Associates Plexiglas boxes (28cm length × 21cm width × 35cm height) encased in sound-attenuating chambers (Med Associates, ST Albans, VT). 10% liquid sucrose (100μL) was delivered through a recessed port located in the center of one wall, containing an infrared beam for detecting port entries and exits. There were two lights (28V, 100mA), one on each side of the port for delivering the 20s continuous light cue, and a house light (28V, 100mA) located at the top of the wall opposite to the port for providing constant background illumination. Next to the house light was a “tweeter” speaker (ENV-224BM) for delivering auditory cues. Footshocks were delivered through the grid floor by a constant current aversive stimulator (ENV-414S). A side-view video camera located on the door of the sound-attenuating chamber recorded the rat’s behavior for offline video analyses.

Estrous cycle monitoring

Estrous phase on the day of the summation test was determined by vaginal swabbing followed by spreading the cells on a microscopic slide. Cells were stained with Cresyl Violet and estrous phase was determined under a light microscope, as in (Shansky et al., 2006). Females were classified as “high estrogen” if in proestrus, and “low estrogen” if in estrus or metestrus. Diestrus was not observed in any of the females on the day of the summation test.

Behavioral conditioning

RFI Cue Discrimination Training

Three stimuli were used as cues: a 20s continuous 3kHz tone (70dB) served as the reward cue, a 20s pulsing 11kHz tone (200ms on, 200ms off; 70dB) as the fear cue, and a 20s continuous light (28V, 100mA) as the safety cue. Stimuli were not counterbalanced in the present study in order to reserve the visual cue as the inhibitor to be presented in compound with one of two auditory tones representing either fear or reward. Our previous studies have not shown any significant differences in the ability to learn reward, fear or safety when auditory and visual stimuli were counterbalanced (Greiner et al., 2019; K H Ng & Sangha, 2022; Sangha et al., 2013).

All 24 male and 24 female rats first received reward pre-training and reward-fear-inhibitor (RFI) cue discrimination training (Figure 1). Reward pre-training consisted of 5 sessions distributed across 5 days. Each session consisted of 25 pairings (ITI, 90–130s) of the reward cue with a 3s delivery of 10% liquid sucrose (100μL pseudorandomly presented 10–20s after reward cue onset) into a port. Rats then received one session of habituation training, which consisted of 25 trials of the reward cue paired with liquid sucrose (100μL pseudorandomly presented 10–20s after reward cue onset), 5 trials of the future fear cue presented alone, and 5 trials of the future safety cue presented alone (ITI, 90–130s). This habituation procedure has been used in this task to assess and reduce any baseline freezing that may be present to the novel cues with the number of trials presented not being sufficient to produce latent inhibition. Rats then received 4 sessions of RFI conditioning across 4 days; i.e. 1 session per day. Each session consisted of 15 trials of the reward cue paired with liquid sucrose (100μL pseudorandomly presented 10–20s after reward cue onset), intermixed with 4 trials of the fear cue paired with footshock (0.5s, 0.5mA at cue offset), and 25 trials of the inhibitor cue presented alone without footshock or sucrose (44 trials total, ITI 100–140s).

Figure 1. Schematic of experimental design.

Figure 1.

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All rats received reward pre-training, discrimination training, and summation tests. Different subsets of rats continued on to assess retardation of fear or reward acquisition. Reward pre-training consisted of a reward cue paired with sucrose delivery (R+). Discrimination training also consisted of interleaved trials of a fear cue paired with footshock (F+) and a inhibitor cue paired with no outcome (I−). The summation test for conditioned inhibition of fear and reward included these same trials as well as compound cues of the reward+inhibitor (RI−) and fear+inhibitory (FI−), both resulting in no outcome. For the retardation of acquisition tests, the inhibitor cue was paired with either footshock or sucrose (I+).

Summation Tests.

All 24 male and 24 female rats underwent a summation test 1 day after RFI4 (Figure 1), which consisted of 5 different types of cued trials. Just like the RFI sessions, the reward cue was paired with liquid sucrose (10 trials; 100μL pseudorandomly presented 10–20s after reward cue onset), intermixed with 4 trials of the fear cue paired with footshock (0.5s, 0.5mA at cue offset), 10 trials of the inhibitor cue presented alone without footshock or sucrose. In addition there were two compound cue trials: the fear and inhibitor cue (FI; 10 trials) presented simultaneously, and the reward and inhibitor cue (RI; 10 trials) presented simultaneously; neither were paired with footshock or sucrose (44 trials total, ITI 100–140s).

Retardation of Acquisition Tests.

Following a summation test, a subset of rats underwent either retardation of fear acquisition (n=6/sex) or retardation of reward acquisition (n=6/sex) (Figure 1). Here, the inhibitor was paired with either footshock for 4 trials or sucrose for 25 trials. The learning curves were compared to the 4 trials of fear+shock in the first discrimination session (RFI1), or to the 25 trials of reward+sucrose in the first reward session (R1), respectively.

Behavioral analyses

Fear behavior was assessed manually offline from videos by measuring freezing, defined as complete immobility with the exception of respiratory movement, which is an innate defensive behavior (Blanchard & Blanchard, 1969; Fendt & Fanselow, 1999). The amount of time spent freezing was quantified and expressed as percentages during the 20s cue presentation, and additionally for the summation test, the 20s pre-cue interval, and the difference between cue and pre-cue. Darting behavior was also assessed due to reports of this being more prominent in female rodents and expressed during fear (Greiner et al., 2019; Gruene et al., 2015; Mitchell et al., 2022). In the current study, if a rat showed at least 1 dart during at least 1 fear trial in the summation test, they were classified as a darter (similar to (Mitchell et al., 2022)). This is different than our previous study (Greiner et al., 2019) where we quantified the number of darts and expressed the data as frequency of darts per cue. In that study, as well as this one, we saw few darts per subject, so instead we showed freezing data disaggregated by darter/nondarter classification. Reward behavior was assessed manually by quantifying the amount of time the animals spent at the port and was expressed as percentages. For the summation test, the 20s pre-cue interval, and the difference between cue and pre-cue were also calculated. Individuals performing the manual behavioral scoring had Pearson’s correlations of at least r = 0.8 with other scorers in the same laboratory for freezing and reward behaviors. Estrous was also monitored in the females, and summation data are shown as a function of high estrogen (high E: proestrus) and low estrogen (low E: estrus, metestrus (diestrus was not observed)).

The behavioral data were analyzed with two-way repeated-measures ANOVAs in GraphPad Prism. For all RFI and summation sessions, freezing to the fear cue was compared (Dunnett’s) to each other cue and reward seeking to the reward cue was compared (Dunnett’s) to each other cue, within sex. Suppression ratios for the summation tests were compared with unpaired t-tests. For the retardation of acquisition sessions, freezing was compared, within sex, across the 4 trials of fear+shock in the first discrimination session, RFI1 (neutral cue) to the 4 trials of inhibitor+shock (inhibitor cue) during the retardation session, and reward seeking was compared, within sex, across the 25 trials of reward+sucrose of the first reward session, R1 (neutral cue) to the 25 trials of inhibitor+sucrose (inhibitor cue) during the retardation session.

Results

Males and females learn to discriminate among reward, fear and inhibitor cues

All 24 male and 24 female rats underwent reward-fear-inhibitor (RFI) cue discrimination training, in which 4 sessions of RFI conditioning occurred across 4 days (1 session per day). Each session consisted of trials of the reward cue paired with liquid sucrose, fear cue paired with footshock, and inhibitor cue presented alone without footshock or sucrose. For freezing behavior (Figure 2A), both males and females showed significant session by cue interactions (Males: F(2.8, 64.2)=29.9, p<0.0001; Females: F(2.5, 48.4) = 11.1, p<0.0001), and main effects of session (Males: F(2.6, 60.6)=28.0, p<0.0001; Females: F(2.5, 56.6) = 10.1, p<0.0001) and cue (Males: F(1.0, 23.2)=577.5, p<0.0001; Females: F(1.0, 23.1) = 1198.0, p<0.0001). Post hoc analyses indicated % time spent freezing during the fear cue was significantly higher than the reward and inhibitor cues in both males and females for all 4 RFI sessions (p<0.0001, each comparison). For port seeking behavior (Figure 2B), both males and females also showed significant session by cue interactions (Males: F(3.3, 76.0) = 4.2, p=0.006; Females: F(3.4, 68.7) = 3.3, p=0.02), and main effects of session (Males: F(1.8, 42.1) = 7.5, p=0.002; Females: F(1.5, 33.4) = 14.0, p=0.0002) and cue (Males: F(1.3, 28.8) = 189.8, p<0.0001; Females: F(1.1, 24.1) = 120.4, p<0.0001). Post hoc analyses indicated % time spent at port during the reward cue was significantly higher than the fear and inhibitor cues in both males and females for all 4 RFI sessions (p<0.0001, each comparison).

Figure 2. Discrimination learning of reward, fear and inhibitor cues.

Figure 2.

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Males (n=24) and females (n=24) learned to discriminate among the fear, reward and inhibitor cues across RFI sessions 1–4. (A) Percentage of time spent freezing to the fear cue was significantly higher than the reward and inhibitor cues across all sessions for both males and females. ****p<0.0001, fear cue compared to reward and inhibitor cues. (B) Percentage of time spent at the port during the reward cue was significantly higher than the fear and inhibitor cues across all sessions for both males and females. ****p<0.0001, reward cue compared to fear and inhibitor cues.

Sex differences in conditioned inhibition of fear, but not reward

All 24 male and 24 female rats also underwent a summation test 1 day after RFI4, which consisted of 5 different types of cued trials. Just like the RFI sessions, there were trials of the reward cue paired with sucrose, fear cue paired with footshock, and inhibitor cue presented alone without footshock or sucrose. In addition there were two compound cue trials: the fear and inhibitor cue (FI) presented simultaneously, and the reward and inhibitor cue (RI) presented simultaneously; neither were paired with footshock or sucrose.

For freezing behavior (Figure 3A), there was a significant cue by sex interaction (F(4, 184) = 3.0, p=0.02), and a main effect of cue (F(1.7, 76.6) = 339.4, p<0.0001), but no effect of sex (F(1,46) = 1.4, p=0.25). Post hoc analyses showed males had significantly higher freezing to the fear cue compared to all other cues (p<0.0001), while females only showed significantly higher freezing to the fear cue compared to the reward, reward+inhibitor, and inhibitor cues (p<0.0001), but not the fear+inhibitor cue (p=0.13). Data for the females were also analyzed by estrous phase on day of the summation test, and shown as a function of high estrogen (high E: proestrus) and low estrogen (low E: estrus, metestrus (diestrus was not observed)). There was no effect of estrous (cue × estrus F(4, 84) = 0.70, p=0.59; estrus F(1, 21) = 1.60, p=0.22; cue F(4, 84) = 128.5, p<0.0001). Within both the males and females there were individual rats that showed darting behavior to at least one fear trial, and are indicated in Figure 3 with dashed lines or open squares. In total, 9/24 males and 11/24 females were classified as darters. Fear suppression ratios (Figure 3B) calculated by taking the % freezing to the FI cue and dividing it by the % freezing to the F cue, showed males had significantly more fear suppression compared to females (unpaired t-test, p=0.02). Again, there was no effect of estrous (high E vs low E; unpaired t-test, p=0.74). Fear suppression ratios within each sex separated by darting was also not significant (male darters vs nondarters, unpaired t-test, p=0.64; female darters vs nondarters, unpaired t-test, p=0.90).

Figure 3. Summation test for conditioned inhibition of fear and reward.

Figure 3.

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During the Summation Test, trials in which the inhibitor was presented concurrently with the reward (RI) and fear (FI) cues were included. (A) Males showed significantly reduced cued freezing to FI compared to F (****p<0.0001, post hoc Dunnett’s), while females did not (p=0.13, post hoc Dunnett’s). This effect did not depend on estrous as females classified as either high estrogen (proestrus) or low estrogen (estrus; metestrus) did not show significant suppression. (B) Suppression ratios of cued freezing to the FI in relation to F were significantly different between males and females (p=0.02, unpaired t-test), indicating males showed greater fear suppression than females. FI/F ratios did not differ in females based on estrous. (C) Freezing during the pre-cue period was significantly higher in females compared to males (**p<0.01, main effect of sex). ALL indicates the average of pre-cue freezing for all trials. (D) The difference of cued freezing minus the pre-cue freezing showed males froze significantly less to FI compared to F (****p<0.0001, post hoc Dunnett’s), while females did not (p=0.46, post hoc Dunnett’s). (E) Males and females showed significantly reduced time at port to RI compared to R (****p<0.0001, post hoc Dunnett’s). There was no effect of estrous (**p<0.01;***p<0.001, post hoc Dunnett’s). (F) Suppression ratios of reward seeking to the RI in relation to R were not significantly different between males and females (p=0.79, unpaired t-test), indicating both males and females showed similar levels of reward suppression. RI/R ratios did not differ in females based on estrous. Dashed lines and open squares are individual rats classified as darters. (G) Time at port during the pre-cue period showed no significant differences between males and females. ALL indicates the average of pre-cue time at port for all trials. (H) The difference of cued port seeking minus the pre-cue port seeking showed males and females reduced port to RI compared to R (****p<0.0001, post hoc Dunnett’s).

Freezing behavior during the 20 s prior to each trial was also quantified, including an average of the pre-cue freezing amongst all trials (Figure 3C), and there was a significant cue by sex interaction (F(5,230) = 2.4, p=0.04), and a main effect of sex (F(1,46) = 7.8, p=0.008), but no effect of cue (F(3.2, 148) = 2.0, p=0.11). Overall, females had higher pre-cue freezing than males. Post hoc analyses (Sidak’s) showed females had significantly higher pre-cue freezing before the fear cue than males (p=0.02). There was a significant interaction of cue by estrous (F(4, 84) = 2.7, p = 0.03), but no main effects of cue (F(2.5, 52.0) = 1.6, p=0.20) or estrous (F(1, 21) = 0.49, p=0.49). Post hoc analyses showed no differences (data not shown).

We also calculated the difference in freezing behavior from pre-cue to cue presentation (% freezing_cue - % freezing_pre-cue) (Figure 3D). Results were similar to the cued freezing analyses: significant cue by sex interaction (F(4, 184) = 3.1, p=0.02), significant main effect of cue (F(2.2, 101.7) = 238.0, p<0.0001), as well as a significant main effect of sex (F(1,46) = 4.3, p=0.04). Post hoc analyses (Dunnett’s) showed males had significantly higher freezing to the fear cue compared to all other cues (p<0.0001), while females only showed significantly higher freezing to the fear cue compared to the reward, reward+inhibitor, and inhibitor cues (p<0.0001), but not the fear+inhibitor cue (p=0.46). Post hoc analyses (Sidak’s) showed no sex differences for any cue. There was a significant interaction of cue by estrous (F(4, 84) = 2.6, p = 0.04), and a main effect of cue (F(2.2, 45.3) = 102.5, p<0.0001), but no main effect of estrous (F(1, 21) = 0.0004, p=0.98). Post hoc analyses (Dunnett’s) showed high estrogen and low estrogen females had higher differential freezing between the fear cue and reward, reward+inhibitor and inhibitor cues (p<0.0001), but not the fear+inhibitor cue (high E, p = 0.21; low E, p = 0.78) (data not shown).

For port seeking behavior (Figure 3E), there was a main effect of cue (F(4, 184) = 81.7, p<0.0001), with no significant cue by sex interaction (F(4, 184) = 0.5, p=0.7) or main effect of sex (F(1, 46) = 0.7, p=0.4). Port seeking was significantly higher during the reward cue compared to all other cues (p<0.0001). Again, data for the females were analyzed by estrous phase on day of the summation test, and shown as a function of high estrogen (high E: proestrus) and low estrogen (low E: estrus, metestrus (diestrus was not observed)). There was no effect of estrous (cue × estrus F(4, 84) = 0.71, p=0.58; estrus F(1, 21) = 0.39, p=0.54; cue F(4, 84) = 27.38, p<0.0001). Reward suppression ratios (Figure 3F) calculated by taking the % port seeking to the RI cue and dividing it by the % port seeking to the R cue, showed no sex difference (unpaired t-test, p=0.79), and no effect of estrous (unpaired t-test, p=0.70).

Port seeking behavior during the 20 s prior to each trial was also quantified, including an average of the pre-cue port amongst all trials (Figure 3G), and there was no significant cue by sex interaction (F(5,230) = 0.41, p=0.84), or main effects of sex (F(1,46) = 2.4, p=0.13) or cue (F(3.0, 137.4) = 0.78, p=0.51). There was also no significant interaction of cue by estrous (F(4, 84) = 0.94, p = 0.45), or main effects of cue (F(2.5, 53.9) = 0.51, p=0.65) or estrous (F(1, 21) = 2.4, p=0.13) (data not shown). We also calculated the difference in port behavior from pre-cue to cue presentation (% port_cue - % port_pre-cue) (Figure 3H). There was no significant cue by sex interaction (F(4, 184) = 0.47, p=0.76) or main effect of sex (F(1, 46) = 3.8, p=0.06), but there was a main effect of cue (F(2.2, 100.1) = 53.0, p<0.0001). Post hoc analyses (Dunnett’s) showed males and females had significantly higher differential port seeking to the reward cue compared to all other cues (p<0.0001). For high versus low estrogen females, there was no significant interaction of cue by estrous (F(4, 84) = 1.13, p = 0.35), or main effect of estrous (F(1, 21) = 3.6, p=0.07), but there was a main effect of cue (F(2.1, 45) = 20.3, p<0.0001). Post hoc analyses (Dunnett’s) showed high estrogen and low estrogen females had higher differential port seeking to the reward cue compared to all other cues (p<0.05) (data not shown).

Retardation of Fear and Reward Acquisition Tests.

During retardation of fear, the inhibitor cue was paired with shock for 4 trials and compared to the 4 fear trials of RFI1 (“neutral cue”) (Figure 4A). Both males and females showed significant trial by cue interactions (Males: F(3, 30) = 3.7, p = 0.02; Females: F(3, 30) = 5.7, p = 0.003), as well as main effects of trial (Males: F(2.1, 21.4) = 24.8, p<0.0001; Females: F(1.8, 18.5) = 51.8, p<0.0001) and cue (Males: F(1, 10) = 8.1, p=0.02; Females: F(1, 10) = 15.0, p=0.003). Freezing was higher for the neutral cue than the inhibitor cue, indicating both males and females showed retardation of fear with the inhibitor cue.

Figure 4. Retardation of acquisition tests for conditioned inhibition of fear and reward.

Figure 4.

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(A) After RFI and summation tests, a subset of animals (6 males, 6 females) received 4 trials of the inhibitor cue paired with footshock. Freezing was compared to the 4 trials of RFI session #1 (initially neutral cue). For both males and females there was less freezing to the inhibitor cue now paired with shock compared to the neutral cue, indicating retardation of fear acquisition to the inhibitor. *p<0.05, **p<0.01, main effects of cue within sex. (B) After RFI and summation tests, a different subset of animals (6 males, 6 females) received 25 trials of the inhibitor cue paired with sucrose. Port seeking was compared to the 25 trials of the first reward session (initially neutral cue). Females, but not males, showed less port seeking to the inhibitor cue now paired with sucrose compared to the neutral cue, indicating retardation of reward acquisition to the inhibitor (**p<0.01, main effect of cue within sex).

During retardation of reward, the inhibitor cue was paired with sucrose for 25 trials and compared to the 25 reward trials of the first reward session, R1 (“neutral cue”) (Figure 4B). In males, there was no significant interaction or main effects found (trial by cue, F(4, 40) = 0.2, p=0.9; trial, F(2.2, 21.5) = 2.7, p=0.1; cue, F(1, 10) = 0.8, p=0.4). Females did show a main effect of cue (F(1, 10) = 18.9, p=0.001) with no significant trial by cue interaction (F(4, 40) = 1.4, p=0.2) or main effect of trial (F(2.2, 22.1) = 1.0, p=0.4). Females showed higher port seeking for the neutral cue than the inhibitor cue, indicating retardation of reward with the inhibitor cue.

Discussion

Here, we used a novel conditioned inhibition of fear and reward paradigm in male and female Long Evans rats where a reward cue was paired with sucrose, a fear cue with foot shock, and an inhibitor cue with neither sucrose or foot shock. During a subsequent summation test for conditioned inhibition of fear and reward, the inhibitor cue was presented concurrently with the reward (RI−) and fear (FI−) cues without any outcome, intermixed with trials of reinforced reward (R+) and fear (F+) cues, and continued nonreinforced trials of the inhibitor alone (I−).

Figure 5 shows a summary of all results of this study. First, males and females showed discriminative reward seeking and fear expression in response to reward, fear and inhibitor cues (Figure 2). During the summation test, males showed conditioned inhibition of freezing to the FI− cue versus the F+ cue, while females did not, which was not dependent on estrous (Figure 3). Males and females did however show conditioned inhibition of reward seeking to the RI− cue versus the R+ cue (Figure 3). During the retardation of fear acquisition test, males and females showed significant retardation of fear acquisition, while only females showed significant retardation of reward acquisition in the retardation of reward acquisition test (Figure 4). Overall, males and females both showed significant reward-fear-inhibitor cue discrimination, conditioned inhibition of reward, and retardation of fear acquisition. The main sex difference, which was found to not be estrous-dependent, was the lack of conditioned inhibition of fear in females to the FI− cue.

Figure 5. Summary of results.

Figure 5.

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For freezing behavior, both males and females showed significant fear discrimination to the RFI cues and retardation of fear acquisition to the inhibitor, while only males showed significant fear suppression during the summation test. For port seeking behavior, both males and females showed significant reward discrimination to the RFI cues and port seeking suppression during the summation test, while only females showed retardation of reward acquisition to the inhibitor. *, indicates significance levels reported in Figs 2–4.

In our prior work, using a different paradigm to study safety, we also observed similar sex differences in conditioned inhibition of freezing (Greiner et al., 2019). In Greiner et al, the summated fear+inhibitor cue was presented throughout all of training alongside the fear, inhibitor and reward cues presented alone. In that study, we did not monitor estrous. Thus, we show once again that females did not significantly downregulate freezing during a summated fear+inhibitor cue compared to the fear cue. We add to this consistent effect by showing it was not dependent on estrous. And, for the first time, we show the same inhibitor cue can be used to assess conditioned inhibition of reward seeking, where both males and females showed a significant downregulation of reward seeking to the summated reward+inhibitor cue compared to the reward cue. Since females also showed retardation of fear and reward acquisition, this indicates the inhibitor cue was gaining inhibitory value in the females. We posit that the reason there was insignificant downregulation of freezing to the FI− cue, is that even though the inhibitor cue gained inhibitory value, the inhibitory value was not great enough to counteract the excitatory value of the F cue driving freezing.

In both this study and our prior study focused on assessing downregulation of fear in males and females (Greiner et al., 2019), we observed darting behavior. In our prior study, darting was almost exclusively seen in the females, but here, in this study, we have an almost equal number of darters within the males (9/24) and females (11/24 subjects). In contrast to our prior study, our current study classified darters as those who displayed at least one dart to at least one fear trial. When darting was observed in any subject, it was typically to the fear trial, and sometimes to the fear+inhibitor trial. In our own laboratory, the observance of darting does seem to vary from study to study, for reasons we are not entirely sure of. One possibility to explain the discrepancy in the current study and our prior study, is that there may have been greater conflict and uncertainty in the current study, since the summated fear+inhibitor cue was only seen during the summation test, whereas in our prior studies we presented it throughout training. That is, in our prior studies, from the very first session with a fear-shock trial there were also summated fear+inhibitor trials without shock, but in the current study the fear/inhibitor conflict was not seen until after RFI training. This could have resulted in higher darting overall, which was particularly noticeable in the males.

Mitchell et al have recently shown differences in the expression of freezing between female darters and female nondarters, with darters showing less expressed freezing (Mitchell et al., 2022). It is important to note that even though their study examined effects of a variety of training factors, such as chamber size and number of fear trials, on the propensity to dart, the authors used a single cue design for auditory fear conditioning; i.e. one auditory cue paired with shock, without any additional cues without shock. In contrast, our task has a higher discriminative demand with not just the inclusion of a CS− (i.e. inhibitor cue), but also a summated CS+/CS− (i.e. fear+inhibitor cue). Our results are not necessarily in conflict with Mitchell et al since we focused on comparing the degree of freezing suppression, and did not focus on absolute levels of freezing. Comparing freezing suppression ratios, which were calculated as % time freezing to the FI− cue divided by % time freezing to the F+ cue, revealed no differences between darters and nondarters within either sex (Figure 3). This is actually consistent with prior reports showing no differences between darters and nondarters in active versus passive coping responses to a single forced swim test after cued fear conditioning (Colom-Lapetina et al., 2019). One interesting observation that came out of our analyses of darters/nondarters was its link to estrus. In our study, most of the female darters (8/11) were in what we referred to as “low E”, which means these females were in either metestrus or estrus on the day of the summation test, and not proestrus. To our knowledge, a relationship between darting behavior and estrus has not been reported before.

Rigorous and historic studies conducted by Rescorla (Rescorla, 1969a, 1969b, 1969c, 1971) led to the establishment of two general tests to assess if a cue has inhibitory properties to act as a conditioned inhibitor. Rescorla argued that for a cue to be considered a valid conditioned inhibitor it needed to pass 2 tests: summation and retardation of acquisition (Rescorla, 1969a). If it only passes one of these, it was proposed that the cue may have either too much or too little attention paid to it, without it gaining inhibitory value. In the first scenario, if a cue passes the summation test but not the retardation of acquisition test, it is thought that too much attention is paid to the cue. For example, significant downregulation of freezing to the FI− may be seen without the delayed fear acquisition to the inhibitor when later paired with shock. In this case, it is thought that there is careful attention to or distraction by the inhibitor without it gaining inhibitory value. In our study, while males passed the summation and retardation tests for fear, they only passed the summation test for reward. When the inhibitor was later paired with sucrose, males did not show a significant difference compared to the original reward cue that was first presented with sucrose. It should be noted though that reward seeking in the males was not very high in that first reward session to the “neutral” cue paired with sucrose (<15% port seeking), and the lack of retardation of reward acquisition in the males may have been due to a trough effect. Females, however, showed ~30% port seeking during initial acquisition (“neutral” cue) which dropped to ~10% port seeking when the inhibitor cue was paired with sucrose. This is in contrast to the freezing behavior to the “neutral” cue paired with shock where both males and females showed 70–80% freezing but only ~40% freezing to the inhibitor cue paired with shock.

In the second scenario, if a cue passes the retardation of acquisition test but not the summation test, it is thought that too little attention is paid to the cue, and may be ignored instead of gaining inhibitory value. Reduced behavior during the retardation test may also be a result of latent inhibition in the current study. The design of the current study presented the inhibitor cue repeatedly without any US throughout training, without compound fear+inhibitor cue presentations. This is unlike our prior work where we presented the fear+inhibitor compound cue, as well as the inhibitor cue alone, throughout training (Greiner et al., 2019; Müller et al., 2018; K. Ng et al., 2023; Ka H Ng et al., 2018; K H Ng & Sangha, 2022; Sangha et al., 2013; Sangha, Greba, et al., 2014; Sangha, Robinson, et al., 2014; Woon et al., 2020). It is thus possible that latent inhibition could have contributed to the slower fear acquisition seen in females to the inhibitor cue when later paired with shock, since significant downregulation of freezing to the FI− was not seen in the summation test. However, when the same inhibitor cue was assessed in the context of conditioned inhibition of reward seeking, the inhibitor passes both tests for conditioned inhibition in the females. Females showed both significant downregulation of reward seeking during RI− versus R+, as well as significantly delayed acquisition of reward seeking when the inhibitor was later paired with sucrose. Together, by having a combined conditioned inhibition of fear and reward paradigm, our results support a view that the inhibitor cue did gain some inhibitory value in the females, perhaps with contributions via latent inhibition mechanisms as well, and the inhibitory strength was enough to pass both tests of conditioned inhibition for reward, but only strong enough to pass the retardation test in conditioned inhibition for fear.

The current study did not assess the influence of context on inhibition as all sessions were conducted in the same context. It is clear from the extinction literature that the inhibitory memory formed during extinction training is limited to the context the extinction training occurred in (Garfinkel et al., 2014; Rougemont-Bücking et al., 2011; Shvil et al., 2014). While a conditioned inhibitor is able to inhibit responding to a target cue across different contexts, both in a fear task (Miguez et al., 2015) and an appetitive task (Bouton & Nelson, 1994), there are some reports that it may be at least partially context-specific. Bouton and Nelson (Bouton & Nelson, 1994) have shown that when a conditioned inhibitor of food trained in context A was tested against the excitatory target in context B, food responding was still inhibited in the new context. However, when the same conditioned inhibitor was trained in two different contexts alongside a unique excitatory stimulus for each context, the conditioned inhibitor was not as effective in reducing food responding to the excitatory stimulus from the other context, indicating the context was influencing inhibitory responding on some level, perhaps serving as an occasion setter in this instance. It is also important to point out that our retardation of acquisition analyses compared responding to the inhibitor paired with shock or sucrose to the original excitor (“neutral” cue). For reward, this was to the reward cue during the very first reward session where they also experienced the context for the first time. For fear, this was to the fear cue in the first discrimination training session, where subjects were more familiar with the context. By the time the inhibitor was paired with either shock or sucrose, all subjects were very familiar with the context, and this may have contributed to “successful” retardation of acquisition. In future, we will include a novel cue to pair with the shock or sucrose during the retardation tests, ideally of the same modality as our current study compared responses to a visual cue to an auditory cue.

It has been previously proposed that an inhibitor trained in an aversive task gains positive valence because it signals the absence of shock, creating relief (De Kleine et al., 2023; Fernando et al., 2014; Rescorla, 1969b), while an inhibitor trained in an appetitive task gains negative valence because it signals the absence of food, creating frustration (Amsel, 1962). Since the males in our study appeared to pass both tests for conditioned inhibition for both aversive and appetitive associations, it does not seem there is any positive or negative valence associated with the inhibitor cue, but instead, in the males, the inhibitor cue represents US omission. Or, the relative strength of the positive and negative valence assigned by the males to the inhibitor cue are equal, resulting in what appears pure expectation of US omission. In the females however, there could be some effects of mixed valence to the inhibitor cue. In addition to signaling US omission, if the females are also assigning negative valence to the inhibitor associated with food omission, without assigning positive valence associated with shock omission, the overall balance would result in the lack of suppressed fear to the FI− cue, while “passing” all the other tests for conditioned inhibition. Together, it suggests that both males and females are appropriately assigning positive and negative valence to the reward/sucrose cue and fear/shock cue, respectively. We also propose that males and females are both learning that the inhibitor cue during RFI training, represents US omission, or “nothing”, but that the inhibitory strength that is learned is greater in males than females, for reasons still to be determined. Then when presented with the summated RI− cue both males and females assign negative valence associated with frustration of the missing food reward, but during the summated FI− cue, only the males are assigning positive valence associated with relief of the missing shock. Ultimately, this would tip the summated balance of males showing suppressed freezing to the FI− cue, but not females.

The question remains as to why the females are not developing as much inhibitory value associated with US omission and/or positive valence associated with shock omission, to the inhibitor cue as males. We do not think it is related to sex differences in shock sensitivity since we previously showed that females were similar to males in measures of jumping and darting in response to shock, and amount of freezing after shock delivery (Greiner et al., 2019). We assessed these measures with shock intensities from 0.35mA to 1.0mA; the current study used 0.5mA as the shock intensity. Even if the females were showing greater shock sensitivity than males, we would expect that not only would the excitatory value of the fear cue increase but also the positive valence associated with shock omission. We also showed here that estrous was not related to the females’ freezing suppression behavior during the summation test. However, we did not assess if estrous at time of first shock (RFI 1) was related to later freezing suppression during the summation test, so that is still a possibility. In our prior work, females tend to show more reward seeking to the reward cue than males (Greiner et al., 2019; Hackleman et al., 2023), which can also be seen in Figure 4B comparing reward seeking to the neutral cue between males and females. However, a big difference between aversive and appetitive conditioning is the number of trials needed to achieve conditioned responding, with appetitive conditioning requiring more trials due to a food reward being less salient then a shock. It is thus possible the excitatory strength of the fear cue is greater than the excitatory strength of the reward cue, leading to significant suppression of reward seeking, but not freezing, in the presence of the inhibitor cue.

As mentioned above, our prior work presented the fear+inhibitor compound cue, as well as the inhibitor cue alone, throughout training (Greiner et al., 2019; Müller et al., 2018; K. Ng et al., 2023; Ka H Ng et al., 2018; K H Ng & Sangha, 2022; Sangha et al., 2013; Sangha, Greba, et al., 2014; Sangha, Robinson, et al., 2014; Woon et al., 2020). This A+, AX− design is more typical in studies of conditioned inhibition (e.g. (Bouton & Nelson, 1994; Laing et al., 2021; Rhodes & Killcross, 2007)). Even with this more typical design, we have shown females do not significantly reduce freezing during the conditioned inhibitor at any point in training (Greiner et al., 2019). Our design is unique in that it incorporates both appetitive and aversive learning while most conditioned inhibition studies include either appetitive or aversive learning. In our prior studies we did not include reward+inhibitor cues to assess conditioned inhibition of reward. In an effort to investigate both conditioned inhibition of fear and reward within animal using one integrated design, we altered our standard discriminative training procedure so that the summated cues did not occur during training, but during a summation test instead. We also used the same stimulus as the inhibitor for both fear and reward. In future, it would be valuable to include unique stimuli trained separately as inhibitors for fear versus reward for comparison in order to tease apart the appetitive and aversive components. In fact, in a recent study where we adapted our rodent conditioned inhibition task to humans, we used separate stimuli for the fear inhibitor and reward inhibitor (Fitzgerald et al., 2023). Our current study also did not counterbalance the auditory and visual stimuli due to practical challenges in creating summated cues (i.e. we could not summate two different tone stimuli). We do know from our prior work, where we counterbalanced the auditory fear cue with the inhibitory light cue, that male and female subjects do not show a difference in learning about the fear or inhibitory cues when it is a tone versus light (Greiner et al., 2019; K H Ng & Sangha, 2022; Sangha et al., 2013). Regardless, in future, using the present paradigm, we will need to improve upon this. It is possible that these procedural differences in training inhibitor cues (i.e. summated cues during training versus test) may have different behavioral and neural mechanisms on how it reduces fear and/or reward seeking behaviors. Using this type of comparative approach across procedures may yield significant advances in understanding inhibitory learning mechanisms, more generally.

Studies on neural circuits governing conditioned inhibition have largely focused on the amygdala, hippocampus and the cortex in both rodents and humans. However, within rodent studies investigating safety in the context of the predictability of threat, areas such as the striatum (Ray et al., 2020; Rogan et al., 2005), periaqueductal gray (Arico et al., 2017; Assareh et al., 2017; Carrive et al., 1997; Vianna et al., 2001; Walker et al., 2020; Wright & McDannald, 2019), paraventricular nucleus of the thalamus, and bed nucleus stria terminalis (Goode et al., 2019, 2020; Ressler et al., 2020) have been shown to contribute. Our own work has identified the infralimbic prefrontal cortex (IL), basolateral amygdala (BLA) and central amygdala (CeA) as key nodes for expressing conditioned inhibition. Briefly, through electrophysiological single unit recordings we found the BLA (Sangha et al., 2013) and IL (K H Ng & Sangha, 2022) have neurons selectively responsive to the fear+inhibitor cue, as well as similarly responsive to fear+inhibitor and reward cues. These latter neurons indicate there is an overlap in learning processes between safety and reward. In the IL, safety-specific and safety/reward overlapping neurons showed neural activity that was negatively correlated with expressed freezing behavior during the fear+inhibitor cue, indicating increased IL activity is correlated with better conditioned inhibition of fear. Inactivating IL neurons projecting to the CeA, via projection-specific chemogenetics, after training sessions that included the fear+inhibitor due prevented expression of conditioned inhibition of fear, while inactivating IL->BLA neurons did not (K. Ng et al., 2023). Using a serial presentation design in which the inhibitor cue was followed by the fear cue, without shock delivery, Falls and Davis (Falls & Davis, 1995) showed the CeA was not necessary for safety acquisition, indicating CeA is contributing more to expression of conditioned inhibition. Meyer et al (Meyer et al., 2019) showed that in both mice and humans ventral hippocampal neurons projecting to the prelimbic region of the prefrontal cortex were more active during the compound presentation of a fear cue and inhibitor cue. The authors hypothesized hippocampal inputs onto local inhibitory neurons within the prelimbic region could be mediating the fear inhibiting effects of the safety cue. This is compatible with other studies, including our own, supporting the general view that conditioned inhibition of fear involves decreased prelimbic activity and increased activity within the infralimbic region of the prefrontal cortex (IL) (Sangha et al., 2020). Much less is known about the neural circuitry of conditioned inhibition of reward but it has been shown that brief pauses in the firing of dopamine neurons in the ventral tegmental area (VTA) are sufficient to create a conditioned inhibitor of reward (Chang et al., 2018). Interestingly, Rhodes and Killcross (Rhodes & Killcross, 2007) showed that while the IL was not required for the summation test in a conditioned inhibition of reward task, it was required to pass the retardation of reward acquisition test. This is in contrast to conditioned inhibition of fear, where our own work has shown the IL is required to pass the summation test (K. Ng et al., 2023; Sangha, Robinson, et al., 2014). Since the task presented in the current study integrates conditioned inhibition of fear and reward, our task would be advantageous to investigate the converging and diverging neural substrates underlying conditioned inhibition of fear versus reward.

Using a variation of the current task, we have shown in healthy humans that skin conductance responses to the FI− cue are correlated with how aversive the subject rated the FI− cue (Fitzgerald et al., 2023). Others have shown that PTSD individuals report increased expectancy of threat following a safety cue (Rabinak et al., 2017), and show persistent fear responses when explicit safety-predictive cues coincide with threat cues, but without threat being present (i.e. FI− cue) (Jovanovic et al., 2009). Patients with anxiety disorders also show higher levels of threat expectancy after a safety cue (De Kleine et al., 2023). Thus, our combined conditioned inhibition task of fear and reward has potentially high translational significance to assess neural underpinnings of inhibitory learning amongst a range of psychiatric disorders.

Acknowledgements

This work was supported by NIMHR01MH110425 to SS. We thank Yolanda Jonker and Signe Hobaugh for excellent animal care.

Footnotes

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