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Contextual fear memory and generalization at proximal time-points (C→A test order), in which the metal grid floor of the training context is retained in the otherwise contextually distinct generalization context. (A) Experimental design and cohort information. (B) Comparison of freezing behavior in male and female mice at 24–48 h after conditioning. (C) Discrimination index. Error bars are mean ± SEM.
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The generalization of fear is adaptive in that it allows an animal to respond appropriately to novel threats that are not identical to previous experiences. In contrast, the overgeneralization of fear is maladaptive and is a hallmark of post-traumatic stress disorder (PTSD), a psychiatric illness that is characterized by chronic symptomatology and...
Similar publications
One strategy to address new potential dangers is to generate defensive responses to stimuli that remind learned threats, a phenomenon called fear generalization. During a threatening experience, the brain encodes implicit and explicit memory traces. Nevertheless, there is a lack of studies comparing implicit and explicit response patterns to novel...
Citations
... These findings suggest that males compared to females show increased generalized fear in adulthood, whether the shock occurred in adolescence or adulthood. This is consistent with other studies showing lower freezing levels in females (Inslicht et al., 2013;Gruene et al., 2015;Colon et al. 2018;Trott et al. 2022; but see Keiser et al. 2017;Lynch et al. 2013) and these sex differences could reflect any number of factors, including how contexts are processed (Asok et al. 2019;Colon and Poulos 2020) or what behaviors are evoked by those contexts (Shanazz et al. 2022;Kirk et al., 1985;Steenbergen et al. 1990;Heinsbroek et al. 1991). ...
Rationale
Intensely stressful experiences can lead to long-lasting changes in appetitive and aversive behaviors. In humans, post-traumatic stress disorder increases the risk of comorbid appetitive disorders including addiction and obesity. We have previously shown that an acute stressful experience in adult male rats suppresses motivation for natural reward.
Objectives
We examine the impact of sex and age on the effects of intense stress on action-based (instrumental) and stimulus-based (Pavlovian) motivation for natural reward (food).
Methods
Rats received 15 unsignaled footshocks (stress) in a single session followed by appetitive training and testing in a distinct context. In Experiment 1, stress occurred in either adolescence (PN28) or adulthood (PN70) with appetitive training and testing beginning on PN71 for all rats. In Experiment 2, stress and appetitive training/testing occurred in adolescence.
Results
Acute stress in adolescent females suppressed instrumental motivation assessed with progressive ratio testing when testing occurred in late adolescence or in adulthood, whereas in males stress in adolescence did not suppress instrumental motivation. Acute stress in adulthood did not alter instrumental motivation. In contrast, Pavlovian motivation assessed with single-outcome Pavlovian-to-instrumental transfer (SO-PIT) was consistently enhanced in females following adolescent or adult stress. In males, however, stress in adolescence had no effect, whereas stress in adulthood attenuated SO-PIT.
Conclusions
Acute stress in adolescence or adulthood altered instrumental motivation and stimulus-triggered Pavlovian motivation in a sex and developmentally specific manner. These findings suggest that the persistent effects of acute stress on Pavlovian and instrumental motivational processes differ in females and males, and that males may be less vulnerable to the deleterious effects of intense stress during adolescence on appetitive motivation.
... The majority of contextual fear generalization investigations in rodents have been performed in males [33], though studies have recently begun to include females. Such sex-inclusive studies indicate that rodents of both sexes can and do exhibit generalized contextual fear, at timepoints both days [55] and weeks [56] after context fear training. Sex differences in the influence of various contextual modalities have been reported (see review [33]), and in our study contextual differences included tactile, olfactory, and visual modalities. ...
A hallmark symptom of many anxiety disorders, and multiple neuropsychiatric disorders more broadly, is generalization of fearful responses to non-fearful stimuli. Anxiety disorders are often comorbid with cardiovascular diseases. One established, and modifiable, risk factor for cardiovascular diseases is salt intake. Yet, investigations into how excess salt consumption affects anxiety-relevant behaviors remains little explored. Moreover, no studies have yet assessed how high salt intake influences generalization of fear. Here, we used adult C57BL/6J mice of both sexes to evaluate the influence of two or six weeks of high salt consumption (4.0% NaCl), compared to controls (0.4% NaCl), on contextual fear acquisition, expression, and generalization. Further, we measured osmotic and physiological stress by quantifying serum osmolality and corticosterone levels, respectively. Consuming excess salt did not influence contextual fear acquisition nor discrimination between the context used for training and a novel, neutral context when training occurred 48 prior to testing. However, when a four week delay between training and testing was employed to induce natural fear generalization processes, we found that high salt intake selectively increases contextual fear generalization in females, but the same diet reduces contextual fear generalization in males. These sex-specific effects were independent of any changes in serum osmolality nor corticosterone levels, suggesting the behavioral shifts are a consequence of more subtle, neurophysiologic changes. This is the first evidence of salt consumption influencing contextual fear generalization, and adds information about sex-specific effects of salt that are largely missing from current literature.
... The reliance on freezing as the primary measure of fear memory is also problematic due to inconsistencies in the literature, where some studies show that females freeze more compared with males [50,55,57,58] and others find similar levels of freezing between the sexes [48,55,[59][60][61][62][63][64]. As above, these findings depend on many experimental factors. ...
... Sex differences are also evident in fear generalization and extinction, which, given similarities between exposure therapy in humans and fear extinction in rodents, could inform the development of sex-specific therapeutics for fear-related disorders. Female rodents generalize fear responses more compared with males, freezing equally in response to both neutral and fearassociated contexts and tones [48,57,58,63,65]. However, increased pre-exposure to the training context [58,66] and testing in the training context before a novel context [58,63] enhance contextual discrimination in female mice, eliminating or reversing sex differences. ...
... Female rodents generalize fear responses more compared with males, freezing equally in response to both neutral and fearassociated contexts and tones [48,57,58,63,65]. However, increased pre-exposure to the training context [58,66] and testing in the training context before a novel context [58,63] enhance contextual discrimination in female mice, eliminating or reversing sex differences. Furthermore, although both sexes extinguish freezing responses when repeatedly presented with a fearassociated context or tone in the absence of footshock, female mice extinguish less effectively than do males when given additional extinction training sessions [67]. ...
Females have historically been disregarded in memory research, including the thousands of studies examining roles for the hippocampus, medial prefrontal cortex, and amygdala in learning and memory. Even when included, females are often judged based on male-centric behavioral and neurobiological standards, generating and perpetuating scientific stereotypes that females exhibit worse memories compared with males in domains such as spatial navigation and fear. Recent research challenges these dogmas by identifying sex-specific strategies in common memory tasks. Here, we discuss rodent data illustrating sex differences in spatial and fear memory, as well as the neural mechanisms underlying memory formation. The influence of sex steroid hormones in both sexes is discussed, as is the importance to basic and translational neuroscience of studying sex differences.
... In addition to potential differences in the expression of contextual fear, there is some evidence that generalization of contextual fear to a novel context is influenced by sex. For instance, Keiser et al. (2017) found greater degrees of generalization in female mice compared to males, and Asok et al. (2019) reported more generalization in female mice tested 3 weeks after initial conditioning, specifically when testing occurred first in the novel context. Further, using step-through avoidance conditioning, Lynch et al. (2013) demonstrated that males and females have equivalent context discrimination when tested early after conditioning. ...
Fear memory retrieval is relevant to psychiatric disorders such as post-traumatic stress disorder (PTSD). One of the hallmark symptoms of PTSD is the repeated retrieval and re-experiencing of the initial fear memory even long after the traumatic event has occurred. Women are nearly twice as likely to develop PTSD following a trauma than men, thus sex differences in the retrieval of fear memories is highly relevant for understanding the development and maintenance of PTSD. In the current study, we aimed to examine sex differences in the retrieval and extinction of either recent or remote fear memories. To do so, we conditioned male and female rats either 1 day (recent) or 28 days (remote) prior to testing retrieval and extinction. While there was no effect of sex or retention interval on initial retrieval, we found that remotely conditioned females exhibited higher rates of freezing than remotely conditioned males in later retrieval/extinction sessions, suggesting a sex difference in the retrieval and/or extinction of remote, but not recent, fear memories. Overall, these results are the first to demonstrate a sex difference in the extinction of remote fear memory, and this may contribute to the differential expression of fear-related disorders like PTSD in men and women.
... (4)(5)(6)(7)). Episodic memories are essential for adaptive behavior and are also the most notably affected in anxiety and post-traumatic stress disorders (PTSD; (8)(9)(10)(11)(12)(13)(14)). Despite considerable progress in identifying how sensory experiences from the real-world are associated with precise biological changes (e.g., ex vivo, in vivo, and at the level of neuronal networks (7,(15)(16)(17)(18)(19)(20)(21)(22)), less is known about which molecules within neuronal networks store information at the biological level. ...
... Moreover, there was a significant correlation with US shock responses and defensive freezing at retrieval and generalization (Fig. 1g-h). Taken together with our recent work on the behavioral mechanisms of fear generalization (8,9), these data suggest fear generalization to complex multisensory environments is likely a product of embedding additional US strong information into the NS○+US weak association in order to produce a NS○+US weak + US strong memory. Furthermore, these data show a strong relationship between the US input parameters (intensity x number), discrete UR motor output type (0-4 ordinal scale; no response flinch, hop, horizontal jump, vertical jump;(80) ), and the later retrieval and expression of an episodic memory via a conditioned freezing response (CR) -a traditional proxy for the strength of a NS○+ US or "CS" memory. ...
... ), along with our recent work on the environmental constraints of recent and remote fear generalization (8,9), we hypothesized that the added US strong information must be separable from all other multisensory noise in the hippocampus at the biological level during the formation of a "strong" episodic memory given the UR / CR relationship above (79). By contrasting animals against the multisensory context-NS○ controls, we could reliably compare the biological bases of US weak to that of US weak + US strong by subtracting out the stochastic multisensory context-NS○ sampling during conditioning. ...
For a generation, neuroscience has searched for a molecule that stores our memories across time. This search has focused on proteomic mechanisms, but less is known about RNA. Here, we identify a new persisting class of RNA associated with long-term memory – Circular RNAs. Unlike other RNAs, Circular RNAs are stable for days or longer and may provide a means for storing sensory information across time. We leveraged a differential fear conditioning paradigm whereby individual mice sample all real-world sensory inputs (i.e., auditory, visual, gustatory, olfactory, and incidental tactile) in a quasi-stochastic manner prior to receiving different intensities of an unconditioned stimulus (US) foot-shock. While Pavlovian models of learning from the 20 th century were critical for understanding elemental associations, they fail to appreciate (1) what US content remains inside of a complex conditioned stimulus (CS) or response (CR – a behavioral manifestation of an episodic memory), (2) what happens when the associations involve multiple senses, and (3) what biologically happens to the real-world US. Given (1) we are constantly sampling information from our environment through all our senses and (2) the US at a given moment in time likely adds value to imprint that multisensory representation, we propose the real-world US is biologically encoded via back-spliced Circular RNAs within the cells and circuits that represent a particular episodic memory and present days later. This logic, best simplified by the equation: , allowed us to ask how the formation of similar episodic memories, which only differ in relation to the content of US information, alter Circular RNAs in the CA1 subfield of the hippocampus – a brain area critical for episodic memories. We found that stronger foot-shock USs during conditioning produce stronger memories relative to weaker USs 24-h later. Stronger memories also generalize to novel/safe environments 48-h later. Moreover, the unconditioned response is highly correlated with future CRs, suggesting (1) an understudied relationship between the strength and type of US/URs and future CRs in complex environments as well as (2) fear generalization, at least in the short-term, is associated with the embedding of additional US information. Next-generation Circular RNA sequencing 1-hr after acquisition revealed a remarkably small set of circular RNAs relative to nearly identical, yet weaker, episodic memories in CA1. Gene Ontologies for mice that formed weaker and stronger memories matched those families classically involved in weaker and stronger forms of memory across species. Preliminary in situ hybridization visually confirmed the presence of Circular RNAs in the CA1 subfield. Future experiments will examine the persistence of Circular RNAs in cells of a memory trace (i.e., engram cells; in situ hybridization) at recent (4 days) and remote (21-days) time points. Taken together with our mathematical model for multisensory learning, our data suggest that Circular RNAs do not contribute to the storage of the multisensory configural representation, but perhaps to the storage of discrete pieces of real-world sensory information related to the US that is partially embedded inside of a memory trace early-on. Importantly, in the above model for multisensory learning, the discrete USs are biologically separable from the future CS (NS+US) associations and US strength is modifiable across time. This work reveals fundamental insights into how we store pieces of real-world sensory information in an episodic memory at the biological level of the brain.
One Sentence Summary
circRNAs biologically encode real-world sensory information into a long-term memory
... In this experiment, we tested each animal in both the shocked (same) context and a novel (different) context in a counterbal-anced fashion. We do not typically do this as results are often difficult to interpret due to testing order effects (Keiser et al. 2017;Asok et al. 2019a), but we wanted to get as much information as possible out of this longer experiment and sought to determine and describe empirically whether order effects would occur. ...
There are sex differences in anxiety disorders with regard to occurrence and severity of episodes such that females tend to experience more frequent and more severe episodes. Contextual fear learning and generalization are especially relevant to anxiety disorders, which are often defined by expressing fear and/or anxiety in safe contexts. In contextual fear conditioning, a representation of the context must first be created, and then that representation must be paired with an aversive consequence. With some variation, the experiments presented here use a 3-d procedure in which day 1 consists of pre-exposure to the to-be-shocked context, day 2 consists of a single context–shock pairing after some placement-to-shock interval (PSI), and day 3 consists of testing in either the same or a novel context. With shorter pre-exposure periods, male rats showed more contextual fear, consistent with previous literature; however, after longer pre-exposure periods, female rats showed greater contextual fear. Additionally, while pre-exposure and PSI are both periods of time prior to the shock, it was found that they were not equivalent to each other. Animals with 120 sec of pre-exposure and a 30-sec PSI show a differential level and time course of fear expression than animals who received no pre-exposure and a 150-sec PSI, and this further depended on sex of the rat. Additionally, an experiment comparing recently versus remotely acquired contextual fear was run. Males were again shown to have greater contextual fear at both time points, and this contextual fear incubated/increased over time in males but not females. To facilitate identification of what processes caused sex differences, we used BaconX, a conceptual and computational model of hippocampal contextual learning. Computational simulations using this model predicted many of our key findings. Furthermore, these simulations suggest potential mechanisms with regard to hippocampal computation; namely, an increased feature sampling rate in males, which may account for the sex differences presented here and in prior literature.
... This finding suggests that there are sex differences when the relationship between acquisition and retrieval is considered and that the acquisition deficit induced by stress can be due to the regulation of emotional response. Our data are consistent with previous results showing sex differences in the performance in rodent memory tasks (Asok et al., 2019;Bangasser et al., 2018;Yokota et al., 2017;Dalla and Shors, 2009). Previously, our group demonstrated that these differences were more robust in tasks involving emotional contexts, including the PMDAT (Ribeiro et al., 2010). ...
Stress encompasses reactions to stimuli that promote negative and positive effects on cognitive functions, such as learning and memory processes. Herein, we investigate the effect of restraint stress on learning, memory, anxiety levels and locomotor activity of male and female mice. We used the plus-maze discriminative avoidance task (PMDAT), a behavioral task based on the innate exploratory response of rodents to new environments. Moreover, this task is used to simultaneously evaluate learning, memory, anxiety-like behavior and locomotor activity. Male and female mice were tested after repeated daily restraint stress (4h/day for 3 days). The results showed stress-induced deficits on aversive memory retrieval only in female mice, suggesting a sexual dimorphism on memory acquisition. Furthermore, stressed females exhibited increased anxiety-like behavior and decreased exploratory behavior. Plasma corticosterone levels were similarly increased by restraint stress in both sexes, suggesting that the behavioral outcome was not related to hormonal secretion. Our findings corroborate previous studies, showing a sexually dimorphic effect of restraint stress on cognition. In addition, our study suggests that stress-related acquisition deficit may be the consequence of elevated emotional response in females.
... .,2018;Asok et al., 2019). Moreover, another study 501 has found no sex-differences in contextual fear discrimi-502 nation(Keeley et al., 2015). ...
In recent years there has been an increase in the development of new synthetic drugs, among which the “bath salt” 3,4-methylenedioxypyrovalerone (MDPV), a psychostimulant with a mechanism of action similar to those of cocaine and amphetamine, stands out. Drugs of abuse have been consistently shown to affect memory function in male rodents. We have recently shown that amphetamine and MDPV induce generalization of fear memory in an inhibitory avoidance discrimination task in male rats. Although abuse of illicit drugs is more prevalent in men than in women, several studies have demonstrated that females are more vulnerable to the effects of drugs of abuse than males and the effects caused by substance dependence on memory in females are still under-investigated. Thus, we examined the effects of subchronic amphetamine or MDPV administrations on memory in a contextual fear conditioning/generalization paradigm in adult male and female rats. Animals were given daily subchronic injections of the drugs, starting 6 days prior the beginning of the behavioral procedures until the end of the paradigm. On day 1 of the experimental protocol, all rats were exposed to a safe context and, the day after, to a slightly different chamber where they received an unsignaled footshock. Twenty-four and forty-eight hours later, freezing behavior and emission of 22 kHz-ultrasonic vocalizations (USVs) were measured in the two different contexts to assess fear memory retention and generalization. Our results indicate that MDPV treatment altered freezing in both sexes, USVs were affected by amphetamine in males while by MDPV in females.
... However, for the experiment in which we assessed fear-potentiated startle, rats were also tested in a context in which shock was not presented, making it akin to a test of contextual discrimination. A number of recent studies (Lynch et al., 2013;Keiser et al., 2017;Asok et al., 2019) have reported that female rodents show a deficit in contextual fear discrimination, where they exhibit higher levels of fear in a novel context compared to males. While our results indicate similar levels of discrimination between sexes, they are not necessarily inconsistent with prior work. ...
... While our results indicate similar levels of discrimination between sexes, they are not necessarily inconsistent with prior work. First, in two of the prior studies (Lynch et al., 2013;Asok et al., 2019) the deficit in discrimination was only seen when the tests occurred several days or more after training. In our study, testing occurred on consecutive days 1 day after training. ...
... In our study, testing occurred on consecutive days 1 day after training. Second, one of the studies (Asok et al., 2019) showed a test order effect such that the deficit in discrimination in females was observed when they were first tested in the novel context, but not if they were tested first in the training context. All rats in our study were first tested in the training context, making it likely that the testing order favored discrimination. ...
The study of fear conditioning has led to a better understanding of fear and anxiety-based disorders such as post-traumatic stress disorder (PTSD). Despite the fact many of these disorders are more common in women than in men, the vast majority of work investigating fear conditioning in rodents has been conducted in males. The goal of the work presented here was to better understand how biological sex affects contextual fear conditioning and expression. To this end, rats of both sexes were trained to fear a specific context and fear responses were measured upon re-exposure to the conditioning context. In the first experiment, male and female rats were given context fear conditioning and tested the next day during which freezing behavior was measured. In the second experiment, rats were trained and tested in a similar fashion while fear-potentiated startle and defecation were measured. We found that males showed more freezing behavior than females during a fear expression test. The expression of fear-potentiated startle did not differ between sexes, while males exhibited more defecation during a test in a novel context. These data suggest that the expression of defensive behavior differs between sexes and highlight the importance of using multiple measures of fear when comparing between sexes.
... Muitos dos estudos que incluem sujeitos do sexo feminino buscam analisar apenas se as fêmeas reproduzem os dados encontrados em machos. Quando não se explora a fundo as especificidades das fêmeas, principalmente quando o comportamento da fêmea difere do comportamento [4] do macho, como em SHAHRIER et al., 2018;ASOK et al., 2019;COLON et al., 2018. Nota-se também que muitos pesquisadores realizam o procedimento de ovariectomia (por exemplo, DÍAZ-VÉLIZ et al., 1997;COST et al., 2012), visando 'eliminar' a variação hormonal, o que resulta em uma avaliação pouco realista dos fenômenos. ...
The female sex has been neglected in neuroscience and behavioral pharmacology research, because of the alleged increased variability in results caused by hormonal fluctuations. In studies with rodents, the vaginal lavage procedure (VLP) is a common method of smear collection to determine the stage of the estrous cycle by cytological analysis. However, little is known about the consequences of this procedure. In neuroscience, spatial memory has been a relevant issue in terms of differences between the sexes. In general, a worse performance is pointed out in females compared to males. However, due to the aforementioned negligence, spatial memory tests were standardized on males. Among the tests that evaluate spatial memory, the task of object location recognition (OLR) allows the evaluation of spatial memory with a character of recognition; the T-maze (TM) task allows the assessment of spatial memory with a referential character and the use of reward; and the plus-maze discriminative avoidance task (PMDAT) allows the concomitant assessment of spatial memory (using aversive stimulation), anxiety-like behavior and locomotion. The aim of the present study was to investigate sex differences and the effects of VLP on the performance of rats in behavioral tasks above. Adult male Wistar rats, adult female Wistar rats, and adult female Wistar rats submitted to 14 days of VLP were evaluated in behavioral tasks. In addition, another set of female rats were submitted to VLP to determine plasma corticosterone levels. Females not submitted to VLP and males had similar performance in the PMDAT task, while females were slightly better in the OLR and TM tasks. Female rats submitted to VLP had impaired performance in the tasks of PMDAT and OLR, but there were no alterations on anxiety-like behavior. The vaginal secretion collection procedure also resulted in a higher level of plasma corticosterone, suggesting that VLP is a stressful manipulation. In conclusion, females do not seem to be naturally worse at spatial memory tasks, and comparative performance in relation to males depends on the type of task and the parameters used in the comparison. Still, the use of VLP to control hormonal variation promotes behavioral and physiological changes per see.
https://repositorio.unifesp.br/handle/11600/61457
