Trace fear conditioning is reduced in the aging rat

Auditory trace fear conditioning is a hippocampus-dependent learning task that requires animals to associate an auditory conditioned stimulus (CS) and a fear-producing shock-unconditioned stimulus (US) that are separated by an empty 20-s trace interval. Previous studies have shown that aging impairs learning performance on hippocampus-dependent tasks. This study measured heart rate (HR) and freezing fear responses to determine if aging impairs hippocampus-dependent auditory trace fear conditioning in freely moving rats. Aging and Young rats received one long-trace fear conditioning session (10 trials). Each trial consisted of a tone-CS (5 s) and a shock-US separated by an empty 20-s trace interval. The next day rats received CS-alone retention trials. Young rats showed significantly larger HR and freezing responses on the initial CS-alone retention trials compared to the Aging rats. A control group of aging rats received fear conditioning trials with a short 1-s trace interval separating the CS and US. The Aging Short-Trace Group showed HR and freezing responses on the initial CS alone retention trials that were similar to the Young Long-Trace Group, but greater than the Aging Long-Trace Group. A second aging control group received unpaired CSs and USs, and showed no HR or freezing responses on CS-alone retention trials. These data show that HR and freezing are effective measures for detecting aging-related deficits in trace fear conditioning.     

Footshock intensity and generalization in contextual and auditory-cued fear conditioning in the rat

The relationship between US (footshock) intensity and the two conditioned freezing responses (to acoustic CS and to "context") was investigated in fear conditioning. Administered footshock intensity was 0.00, 0.15, 0.30, 0.60, 0.90, and 1.20 mA to six different groups of 70-day-old male Albino Wistar rats. To measure contextual freezing, the animals were again placed inside the conditioning apparatus without acoustic CS and US presentation. To measure acoustic CS freezing, the animals were placed in a totally different apparatus and only the acoustic CS was presented. The 0.15 mA footshock intensity was not sufficient to condition the animals, in fact no freezing was exhibited as in the non-shocked control group. The 0.30 mA footshock intensity was sufficient only to condition the animals to the acoustic CS, whereas the 0.60 mA was sufficient to condition the animals both to acoustic CS and to context. Footshock intensities (0.90 and 1.20 mA) did not elicit any significant increase in conditioned freezing for either acoustic CS or context but at the highest one the generalization phenomenon appeared (freezing in the different context before presentation of acoustic CS). Acoustic CS freezing to all over-threshold intensities was longer than that to context. In conclusion, freezing responses to acoustic CS and context after increasing footshock intensities follow distinct patterns, and intermediate footshock intensities (0.60 and 0.90 mA) appear to be the most useful for eliciting conditioned freezing responses to acoustic CS and to context without inducing a generalized fear status contamination.     

Aversive conditioning in the rat: effects of a benzodiazepine and of an opioid agonist and antagonist on conditioned hypoalgesia and fear.

Hypoalgesia and fear co-occurred in rats trained on a heated floor and tested for their latencies to paw lick on that floor and to step down onto a nonheated floor. These responses were extinguished, suggesting a mediation by aversive conditioning processes. A benzodiazepine impaired the acquisition of aversive conditioning, but it did not attenuate the expression of conditioned hypoalgesia. The opioid agonist morphine also impaired acquisition across a range of drug doses and variations in hypoalgesic tolerance, whereas the opioid antagonist naloxone facilitated acquisition. The results are discussed in terms of the perceptual-defensive-recuperative (Fanselow, 1986) and working memory (Grau, 1987) models of the mechanisms for the co-occurrence of conditioned hypoalgesia and fear.     

Genetic background differences and nonassociative effects in mouse trace fear conditioning

Fear conditioning, including variants such as delay and trace conditioning that depend on different neural systems, is widely used to behaviorally characterize genetically altered mice. We present data from three strains of mice, C57/BL6 (C57), 129/SvlmJ (129), and a hybrid strain of the two (F(1) hybrids), trained on various versions of a trace fear-conditioning protocol. The initial version was taken from the literature but included unpaired control groups to assess nonassociative effects on test performance. We observed high levels of nonassociative freezing in both contextual and cued test conditions. In particular, nonassociative freezing in unpaired control groups was equivalent to freezing shown by paired groups in the tests for trace conditioning. A number of pilot studies resulted in a new protocol that yielded strong context conditioning and low levels of nonassociative freezing in all mouse strains. During the trace-CS test in this protocol, freezing in unpaired controls remained low in all strains, and both the C57s and F(1) hybrids showed reliable associative trace fear conditioning. Trace conditioning, however, was not obtained in the 129 mice. Our findings indicate that caution is warranted in interpreting mouse fear-conditioning studies that lack control conditions to address nonassociative effects. They also reveal a final set of parameters that are important for minimizing such nonassociative effects and demonstrate strain differences across performance in mouse contextual and trace fear conditioning.     

Anterograde amnesia for Pavlovian fear conditioning and the role of one-trial overshadowing: effects of preconditioning exposures to morphine in the rat

Four experiments studied anterograde deficits in Pavlovian fear conditioning following prolonged exposure to the mu-opioid receptor agonist morphine. Injections of morphine produced temporally graded anterograde amnesia characterized by deficits in contextual and conditioned-stimulus (CS) conditioning 1 or 7 days and selective impairment in CS conditioning 21 days after last injection. This anterograde deficit in conditioning did not recover across a retention interval, was absent when rats were tested immediately after conditioning, and required the presence of an auditory CS. These results suggest that anterograde deficits in Pavlovian fear conditioning emerged from differences in susceptibility to 1-trial overshadowing of context by CS.     

Overt behavior and ultrasonic vocalization in a fear conditioning paradigm: a dose-response study in the rat

The behavioral analysis of laboratory rats is usually confined to the level of overt behavior, like locomotion, behavioral inhibition, instrumental responses, and others. Apart from such visible outcome, however, behaviorally relevant information can also be obtained when analyzing the animals' ultrasonic vocalization, which is typically emitted in highly motivational situations, like 22-kHz calls in response to acute or conditioned threat. To further investigate such vocalizations and their relationship with overt behavior, we tested male Wistar rats in a paradigm of Pavlovian fear conditioning, where a tone stimulus (CS) was preceding an aversive foot-shock (US) in a distinct environment. Importantly, the shock dose was varied between groups (0-1.1 mA), and its acute and conditioned outcome were determined. The analysis of visible behavior confirms the usefulness of immobility as a measure of fear conditioning, especially when higher shock doses were used. Rearing and grooming, on the other hand, were more useful to detect conditioned effects with lower shock levels. Ultrasonic vocalization occurred less consistently than changes in overt behavior; however, dose-response relationships were also observed during the phase of conditioning, for example, in latency, call rate and lengths, intervals between calls, and sound amplitude. Furthermore, total calling time (and rate) were highly correlated with overt behavior, namely behavioral inhibition as measured through immobility. These correlations were observed during the phase of fear conditioning, and the subsequent tests. Importantly, conditioned effects in overt behavior were observed, both, to the context and to the CS presented in this context, whereas conditioned vocalization to the context was not observed (except for one rat). In support and extent of previous results, the present data show that a detailed analysis of ultrasonic vocalization can substantially broaden and refine the spectrum of analysis in behavioral work with rats, since it can provide information about situational-, state-, and subject-dependent factors which are partly distinct from what is visible to the experimenter.     

The role of the nucleus basalis magnocellularis in fear conditioning consolidation in the rat

The nucleus basalis magnocellularis (NBM) is known to be involved in the memorization of several conditioned responses. To investigate the role of the NBM in fear conditioning memorization, this neural site was subjected to fully reversible tetrodotoxin (TTX) inactivation during consolidation in adult male Wistar rats that had undergone fear training to acoustic conditioned stimulus (CS) and context. TTX was stereotaxically administered to different groups of rats at increasing intervals after the acquisition session. Memory was assessed as the conditioned freezing duration measured during retention testing, always performed 72 and 96 h after TTX administration. In this way, there was no interference with normal NBM function during either acquisition or retrieval phases, allowing any amnesic effect to be due only to consolidation disruption. The results show that for contextual fear response memory consolidation, NBM functional integrity is necessary up to 24 h post-acquisition. On the other hand, NBM functional integrity was shown to be necessary for memory consolidation of the acoustic CS fear response only immediately after acquisition and not 24-h post-acquisition. The present findings help to elucidate the role of the NBM in memory consolidation and better define the neural circuits involved in fear memories.     

Olfactory fear conditioning induces field potential potentiation in rat olfactory cortex and amygdala

The widely used Pavlovian fear-conditioning paradigms used for studying the neurobiology of learning and memory have mainly used auditory cues as conditioned stimuli (CS). The present work assessed the neural network involved in olfactory fear conditioning, using olfactory bulb stimulation-induced field potential signal (EFP) as a marker of plasticity in the olfactory pathway. Training consisted of a single training session including six pairings of an odor CS with a mild foot-shock unconditioned stimulus (US). Twenty-four hours later, the animals were tested for retention of the CS as assessed by the amount of freezing exhibited in the presence of the learned odor. Behavioral data showed that trained animals exhibited a significantly higher level of freezing in response to the CS than control animals. In the same animals, EFPs were recorded in parallel in the anterior piriform cortex (aPC), posterior piriform cortex (pPC), cortical nucleus of the amygdala (CoA), and basolateral nucleus of the amygdala (BLA) following electrical stimulation of the olfactory bulb. Specifically, EFPs recorded before (baseline) and after (during the retention test) training revealed that trained animals exhibited a lasting increase (present before and during presentation of the CS) in EFP amplitude in CoA, which is the first amygdaloid target of olfactory information. In addition, a transient increase was observed in pPC and BLA during presentation of the CS. These data indicate that the olfactory and auditory fear-conditioning neural networks have both similarities and differences, and suggest that the fear-related behaviors in each sensory system may have at least some distinct characteristics.     

Classical fear conditioning in the anxiety disorders: a meta-analysis

Fear conditioning represents the process by which a neutral stimulus comes to evoke fear following its repeated pairing with an aversive stimulus. Although fear conditioning has long been considered a central pathogenic mechanism in anxiety disorders, studies employing lab-based conditioning paradigms provide inconsistent support for this idea. A quantitative review of 20 such studies, representing fear-learning scores for 453 anxiety patients and 455 healthy controls, was conducted to verify the aggregated result of this literature and to assess the moderating influences of study characteristics. Results point to modest increases in both acquisition of fear learning and conditioned responding during extinction among anxiety patients. Importantly, these patient-control differences are not apparent when looking at discrimination studies alone and primarily emerge from studies employing simple, single-cue paradigms where only danger cues are presented and no inhibition of fear to safety cues is required.     

Impact of hormonal contraceptives on sex differences in fear conditioning and fear extinction in PTSD

Sex differences in the neurobiological mechanisms involved in fear conditioning and extinction have been suggested to contribute to differential vulnerability for the development of posttraumatic stress disorder (PTSD) in women compared with men. Reproductive hormones, such as estradiol, have been shown to facilitate fear conditioning and extinction learning and may explain some of these differences. However, the effect of commonly used hormonal contraceptives on the neurobiological mechanisms of fear conditioning and extinction is poorly understood. A laboratory study was conducted in trauma-exposed men and women with and without full or partial PTSD to examine effects of sex and use of hormonal birth control on fear conditioning, fear extinction learning, and extinction retention. Participants underwent fear conditioning with stimuli that were paired (CS+) or unpaired (CS−) with shock. Extinction learning occurred 72 h later, and extinction retention was tested 1 wk after extinction. Women on hormonal contraceptives (HCs) demonstrated enhanced acquisition of fear conditioning and enhanced extinction of fear as compared with women off hormonal birth control and men. While clinical implications have yet to be determined, these results suggest that hormonal contraceptives may facilitate learning during both fear acquisition and extinction. Understanding the impact of sex and hormones on fear conditioning and extinction processes may lead to new insights into the pathophysiology of PTSD and result in advancements in treatment that may vary by sex.

It is widely recognized that posttraumatic stress disorder (PTSD) is a common consequence of trauma exposure, and women are at particularly high risk, with some but not all studies finding that women develop PTSD at twice the rate of men, despite greater trauma exposure in men (Breslau et al. 1998; Tanielian et al. 2008). Although some have suggested that greater exposure to interpersonal violence may contribute to higher rates of PTSD in women, other evidence implicates sex differences in the neurobiological mechanisms that are involved in fear conditioning and extinction. Enhanced fear conditioning and diminished extinction of conditioned fear have been associated with higher levels of endogenous estrogen in women, as well as the development and maintenance of PTSD in both sexes (Orr et al. 2000; Milad et al. 2009b; Glover et al. 2012). Furthermore, as one of the most empirically supported treatments for PTSD is prolonged exposure therapy, which is largely based on fear extinction principles and the success of extinction learning (Rothbaum and Davis 2003), a clear understanding of the individual factors impacting fear conditioning and extinction is critical.Although some of the studies examining sex differences in fear conditioning are inconsistent (Guimaraes et al. 1991; Zorawski et al. 2005; Milad et al. 2006), an increasing body of evidence from rodent and human studies supports the existence of sex differences in fear extinction learning and recall (Maren et al. 1994; Pryce et al. 1999; Milad et al. 2009a; Merz et al. 2013). One possible explanation for the lack of consistent findings in the fear conditioning literature may be related to potential floor effects associated with subclinical impairment of nonclinical samples, as most laboratory studies examining sex differences were conducted in healthy humans. Another explanation that is gaining substantial support is the impact of hormones that differ between the sexes, among individuals, and even within individuals across time (Quirk and Mueller 2008; Lebron-Milad and Milad 2012; Arevalo et al. 2015; Hwang et al. 2015; Herrera et al. 2017; Maeng et al. 2017; Antov and Stockhorst 2018).The available literature indicates that estradiol, the primary estrogen in women during the childbearing years, is also present at overall lower concentrations in males and plays a large role in the fear conditioning and extinction differences observed between men and women (Gupta et al. 2001; Jasnow et al. 2006; Chang et al. 2009; Milad et al. 2009a, 2010; Zeidan et al. 2011; Maddox et al. 2018; Matsumoto et al. 2018; Carvalho et al. 2021). In both sexes, estradiol plays a variety of important functions in the brain, including the regulation of oxidative stress, inflammation, and gene expression, as well as in cognitive functions such as learning and memory (Hammoud et al. 2020). Estrogen receptors are found throughout brain regions that are important for fear conditioning and extinction processes (e.g., the amygdala, ventromedial prefrontal cortex, and hippocampus), likely via enhancements to learning and memory (Milad et al. 2008; Quirk and Mueller 2008; Lebron-Milad and Milad 2012). In women, as peripheral levels of estradiol vary over the course of the menstrual cycle, so do levels of estradiol in the brain (Arevalo et al. 2015). Estradiol has been shown to enhance memory consolidation across stages of fear conditioning, extinction, and retention (Lebron-Milad and Milad 2012). Most relevant to learning in PTSD, women with high levels of estradiol demonstrate enhanced memory formation in the presence of stress exposure (Herrera et al. 2017; Antov and Stockhorst 2018). In animal and human models, higher estradiol levels appear to facilitate acquisition of fear conditioning and extinction (Maeng et al. 2017). For example, women with high endogenous estradiol levels have enhanced responses in fear circuitry during fear conditioning, extinction, and recall as compared with men (Hwang et al. 2015). When phase of menstrual cycle has been taken into account, differences have been observed in conditioned fear responses and severity of PTSD symptoms. Specifically, when women are in the midluteal phase of the menstrual cycle (higher endogenous estradiol), they demonstrate a stronger positive relationship between SCR during fear conditioning and PTSD symptoms than women in the early follicular phase of menstruation (lower endogenous estradiol) (Carpenter et al. 2022). In the complimentary literature on the startle response, low-estradiol women demonstrated reduced discrimination between CS+ and CS− during fear conditioning and reduced inhibition of fear-potentiated startle during extinction and extinction recall, indicating less successful learning than their high-estradiol counterparts (Glover et al. 2012, 2013; Armbruster et al. 2018). This literature suggests that higher levels of estradiol relate to enhanced acquisition of associations between an unconditioned stimulus (UCS) and a conditioned stimulus (CS) during fear conditioning and enhanced extinction of this association during the extinction phase due to greater memory consolidation.A variety of factors can account for hormone differences in women, including menstrual phase, age, and use of hormonal birth control. Approximately 11%–20% of women aged 20–39 yr use oral contraceptives (OCs) (Daniels and Abma 2020). Commonly used OCs directly affect estradiol levels and hormonal fluctuation associated with the menstrual cycle. However, hormonal contraceptives (HCs) have received little attention in the fear conditioning literature. At the time of writing, we were unable to locate studies examining the impact of other HCs on fear conditioning circuitry, although these forms of birth control are increasingly popular among women, and evidence suggests that a variety of HCs has impacts on brain structure, function, and cognitive processes (Brønnick et al. 2020). Literature suggests that estradiol levels in women on HCs are typically low, similar to those of women in the early follicular phase of menstruation (Brynhildsen 2014). Although Hwang et al. (2015) did not demonstrate an effect of HCs on fear conditioning, many HCs contain ethinyl estradiol, which is synthetic estrogen that binds to estrogen receptors at high levels. Further research is needed to determine whether synthetic estrogen present in HCs impacts fear conditioning and fear extinction and contributes to associated sex differences, particularly in a highly sensitized population such as those with PTSD. In the current study, differences in fear conditioning, extinction, and retention were examined in women on hormonal birth control and in the early follicular phase of the menstrual cycle as compared with men to determine whether the synthetic hormones present in HCs confer any enhancement to these processes over and above women off of birth control with theoretically low levels of endogenous estradiol.Fear conditioning is measured by assessing the differential SCR to a conditioned stimulus (CS+) paired with an unconditioned stimulus (shock; UCS) and a stimulus unpaired with a shock (CS−). Greater acquisition of fear conditioning is evidenced by greater SCR response to the CS+ when compared with the CS−. Fear extinction refers to repeated exposure to the CS in the absence of the US, which results in diminishing reactivity to previously conditioned stimuli due to an inhibitory neural link that is formed (Myers and Davis 2007). Extinction is therefore operationalized by reduced discrimination of responding to the CS+ and CS− over time, and its retention is evidenced by a maintenance of low differential SCR in response to presentation of CS+ and CS− cues at follow-up.In previous work, our group examined sex differences in skin conductance responses to a fear conditioning paradigm in men and women with PTSD (Inslicht et al. 2013). In that sample, women were premenopausal and underwent conditioning during the follicular phase of the menstrual cycle. We found that women had greater differential fear acquisition compared with men. Other work has indicated that the effects of endogenous gonadal hormones on fear extinction are moderated by PTSD diagnosis, such that women with PTSD demonstrated impaired fear extinction during the midluteal phase of menstruation but not during the early follicular phase (Pineles et al. 2016b). The current study examines the effect of sex, HC use, and PTSD severity on fear conditioning, fear extinction, and extinction retention in medically healthy trauma-exposed premenopausal women on or off HCs and age-matched men across a range of PTSD symptom severity. We used a validated laboratory conditioning paradigm (Inslicht et al. 2021) that occurred over several days in which fear acquisition was separated from extinction by 72 h to avoid influencing consolidation of fear conditioning, and extinction retention was evaluated 1 wk after the fear extinction session to provide a test of durability of extinction over time.Given evidence for impaired fear extinction in PTSD, we hypothesized that participants high in PTSD (men and women combined) would have decreased fear extinction learning and extinction retention compared with those with low PTSD symptom scores. As estradiol appears to enhance learning during stress in women, we predicted that women on HCs would demonstrate higher differential SCR during acquisition and lower differential SCR during extinction and extinction retention than naturally cycling women in the early follicular phase of menstruation and men. Finally, we predicted a PTSD × sex interaction effect for extinction learning and retention; women on HCs with high levels of current PTSD would have enhanced acquisition but decreased extinction learning and retention compared with women with low levels of current PTSD and men.     

The effects of distributed training on retention of operant conditioning in human infants

Long-term retention of operant footkicking acquired in the mobile conjugate reinforcement paradigm was assessed as a function of the distribution of training time. In the first study, 3-month-old infants were trained for either one 18-min session or for two 9-min or three 6-min sessions separated by 24-hr intervals. All infants exhibited retention during a test administered immediately after training, but only those trained in a single session continued to perform the conditioned response during cued-recall tests 7 or 14 days later. Infants trained in three sessions showed no evidence of remembering the contingency even after a week. A warm-up decrement, seen in the day-to-day performance of infants in the distributed conditions, was eliminated in the second study by the interpolation of a nonreinforcement period at the outset of daily sessions. This procedure also enhanced long-term retention such that infants trained in three 6-min session now remembered the contingency for 14 days and did not differ from infants who had received a single 18-min session. Whether distributed training facilitates or impairs long-term retention appears to depend on the opportunity for infants to acquire a sufficient number (or kind) of effective retrieval cues during original learning.     

Classical conditioning and retention of the infant's eyelid response: effects of age and interstimulus interval

Independent groups of 10-,20-, and 30-day-old infants were subjected to a classical eyelid conditioning procedure involving either a 500- or a 1500-msec interstimulus interval (ISI). Ten days later, all received a second conditioning session. A reliable increase in conditioned responding was observed at all ages but only by infants receiving the 1500-msec ISI. Although age was not a significant factor in any conditioning measure except final performance level, which was greater for the oldest than for the youngest group, it did influence long-term retention. A reliable memory component was observed in the Session 2 performance of infants initially trained at 20 and 30 days but not at 10 days. These data demonstrate the importance of temporal parameters in the formation of conditioned associations very early in infancy and provide evidence for the long-term behavioral consequences of those associations.