Effects of group size on movement patterns and clustering dynamics in rats.

Environment is a determining factor that can facilitate or hinder social interactions. A precursor to meaningfully engaging with conspecifics is being exposed to opportunistic encounters with others. Increasing the number of individuals in a given space (thus increasing density) would, statistically speaking, increase the likelihood of accidental encounters. This might have consequences on the formation of social networks-an idea that has not reliably been explored. If true, we would expect that increasing density would lead to an increase in the number and the duration of 'clusters' of animals. Here, we examined whether varying the number of rats in an open field environment differentially affected their movement dynamics or their propensity to aggregate into clusters and, if so, whether such effects are dependent solely on statistical factors due to increases in density, the potential for actively-sought social interactions, or both. We found that the number of rats in an environment impacts ambulation speed, distance traveled, cluster formation and approaches, and that number and duration of clusters are highly dependent on the propensity for the rats to engage in social interactions.

Conspecific interactions predict social transmission of fear in female rats.

Social transmission of fear occurs in a subset of individuals, where an Observer displays a fear response to a previously neutral stimulus after witnessing or interacting with a conspecific Demonstrator during memory retrieval. The conditions under which fear can be acquired socially in rats have received attention in recent years, and suggest that social factors modulate social transmission of information. We previously found that one such factor, social rank, impacts fear conditioning by proxy in male rats. Here, we aimed to investigate whether social roles as determined by nape contacts in females, might also have an influence on social transmission of fear. In-line with previous findings in males, we found that social interactions in the home cage can provide insight into the social relationship between female rats and that these relationships predict the degree of fear acquired by-proxy. These results suggest that play behavior affects the social transfer/transmission of information in female rats.

Carbon Dioxide Reactivity Differentially Predicts Fear Expression After Extinction and Retrieval-Extinction in Rats.

Background: Cues present during a traumatic event may result in persistent fear responses. These responses can be attenuated through extinction learning, a core component of exposure therapy. Exposure/extinction is effective for some people, but not all. We recently demonstrated that carbon dioxide (CO2) reactivity predicts fear extinction memory and orexin activation and that orexin activation predicts fear extinction memory, which suggests that a CO2 challenge may enable identification of whether an individual is a good candidate for an extinction-based approach. Another method to attenuate conditioned responses, retrieval-extinction, renders the original associative memory labile via distinct neural mechanisms. The purpose of the current study was to examine whether we could replicate previous findings that retrieval-extinction is more effective than extinction at preventing the return of fear and that CO2 reactivity predicts fear memory after extinction. We also examined whether CO2 reactivity predicts fear memory after retrieval-extinction. Methods: Male rats first underwent a CO2 challenge and fear conditioning and were assigned to receive either standard extinction (n = 28) or retrieval-extinction (n = 28). Then, they underwent a long-term memory (LTM) test and a reinstatement test. Results: We found that retrieval-extinction resulted in lower freezing during extinction, LTM, and reinstatement than standard extinction. Using the best subset approach to linear regression, we found that CO2 reactivity predicted LTM after extinction and also predicted LTM after retrieval-extinction, although to a lesser degree. Conclusions: CO2 reactivity could be used as a screening tool to determine whether an individual may be a good candidate for an extinction-based therapeutic approach.

Extinction learning underlies exposure therapy, a treatment for anxiety disorders. However, not everyone benefits from exposure therapy, highlighting the need in developing approaches that may help predict which individuals will respond. We tested whether extinction or an alternative treatment called retrieval-extinction would be more effective at reducing conditioned fear responses in rats and whether the response to a carbon dioxide (CO₂) challenge would predict treatment response. We found that retrieval-extinction was more effective at reducing fear, and CO₂ reactivity was better at predicting the response to extinction. These findings could help improve treatment strategies for anxiety disorders.

Reconsolidation and Fear Extinction: An Update.

Fear memories can be updated behaviorally by delivering extinction trials during the reconsolidation window, which results in a persistent attenuation of fear memories (Monfils et al., Science 324:951-955, 2009). This safe and non-invasive paradigm, termed retrieval-extinction (or post-retrieval extinction), has also been found to be successful at preventing the return of fear in healthy fear conditioned humans (Schiller et al., Nature 463:49-53, 2010), and in the time since its discovery, there has been an explosion of research on the use of retrieval-extinction in fear memories in humans and other animals, some of which have found a long-term reduction in conditioned responding, and some who have not. These discrepant findings have raised concerns as to whether retrieval-extinction really results in updating of the original fear memory, or if it simply enhances extinction. We will first review the progress made on elucidating the cellular mechanisms underlying the fear attenuating effects of retrieval-extinction and how they differ from traditional extinction. Special attention will be paid to the molecular events necessary for retrieval-extinction to successfully occur and how these reconsolidated memories are represented in the brain. Next, we will examine the parameters that determine whether or not a memory will be updated via extinction during the reconsolidation window (also known as boundary conditions). These boundary conditions will also be discussed as possible explanations for discrepant findings of the retrieval-extinction effect. Then we will examine the factors that can determine whether an individual's fears will successfully be attenuated by retrieval-extinction. These individual differences include genetics, age, and psychopathology. Finally, we will discuss recent attempts to bring the retrieval-extinction paradigm from the bench to the bedside for the behavioral treatment of anxiety and trauma disorders.

Estrous cycle state-dependent renewal of appetitive behavior recruits unique patterns of Arc mRNA in female rats.

Introduction: Renewal is a behavioral phenomenon wherein extinction learning fails to generalize between different contextual environments, thereby representing a significant challenge to extinction-based rehabilitative therapies. Previously, we have shown that renewal of extinguished appetitive behavior differs across the estrous cycle of the female rat. In this experiment that effect is replicated and extended upon to understand how the estrous cycle may modulate contextual representation at the neuronal population level to drive renewal. Methods: Estrous cycle stage [i.e., proestrus (P, high hormone) or metestrus/diestrus (M/D, low hormone)] was considered during two important learning and behavioral expression windows: at extinction training and during long-term memory (LTM)/renewal testing. Cellular compartment analysis of temporal activity using fluorescence in situ hybridization (catFISH) for Arc mRNA was conducted after the distinct context-stimulus exposures. Results: Rats in P during context-dependent extinction training but in a different stage of the estrous cycle during LTM and renewal testing (P-different) were shown to exhibit more renewal of conditioned foodcup (but not conditioned orienting) behavior compared to rats in other estrous cycle groups. Importantly, we discovered this depends on the order of tests. P-different rats showed differential Arc mRNA expression in regions of the prefrontal cortex (PFC), amygdala, and hippocampus (HPC). For each case P-different rats had more co-expression (i.e., expression of both nuclear and cytoplasmic) of Arc mRNA compared to other groups; specific to the dorsal HPC, P-different rats also had a more robust Arc mRNA response to the extinction context exposure. Conclusion: These data suggest female rats show estrous cycle state-dependent renewal of appetitive behavior, and differences in context and conditioned stimulus representation at the neuronal level may drive this effect.

Observing a trained demonstrator influences associative appetitive learning in rats.

The ability to acquire information about the environment through social observation or instruction is an essential form of learning in humans and other animals. Here, we assessed the ability of rats to acquire an association between a light stimulus and the presentation of a reward that is either hidden (sucrose solution) or visible (food pellet) via observation of a trained demonstrator. Subsequent training of observers on the light-reward association indicated that while observation alone was not sufficient for observers to acquire the association, contact with the reward location was higher in observers that were paired with a demonstrator. However, this was only true when the light cue predicted a sucrose reward. Additionally, we found that in the visible reward condition, levels of demonstrator orienting and food cup contact during the observation period tended to be positively correlated with the corresponding behaviour of their observer. This relationship was only seen during later sessions of observer training. Together, these results suggest that while our models were not sufficient to induce associative learning through observation alone, demonstrator behaviour during observation did influence how their paired observer's behavioural response to the cue evolved over the course of direct individual training.

CO2 reactivity is associated with individual differences in appetitive extinction memory.

Pavlovian conditioning can underly the maladaptive behaviors seen in psychiatric disorders such as anxiety and addiction. In both the lab and the clinic, these responses can be attenuated through extinction learning, but often return with the passage of time, stress, or a change in context. Extinction to fear and reward cues are both subject to these return of behavior phenomena and have overlap in neurocircuitry, yet it is unknown whether they share any common predictors. The orexin system has been implicated in both fear and appetitive extinction and can be activated through a CO2 challenge. We previously found that behavioral CO2 reactivity predicts fear extinction and orexin activation. Here, we sought to extend our previous findings to determine whether CO2 reactivity might also predict extinction memory for appetitive light-food conditioning. We find that the same subcomponent of behavioral CO2 reactivity that predicted fear extinction also predicts appetitive extinction, but in the opposite direction. We show evidence that this subcomponent remains stable across two CO2 challenges, suggesting it may be a stable trait of both behavioral CO2 reactivity and appetitive extinction phenotype. Our findings further the possibility for CO2 reactivity to be used as a transdiagnostic screening tool to determine whether an individual would be a good candidate for exposure therapy.

Patterns of Arc mRNA expression in the rat brain following dual recall of fear- and reward-based socially acquired information.

Learning can occur via direct experience or through observation of another individual (i.e., social learning). While research focused on understanding the neural mechanisms of direct learning is prevalent, less work has examined the brain circuitry mediating the acquisition and recall of socially acquired information. Here, we aimed to further elucidate the mechanisms underlying recall of socially acquired information by having male and female rats sequentially recall a socially transmitted food preference (STFP) and a fear association via fear conditioning by-proxy (FCbP). Brain tissue was processed for mRNA expression of the immediate early gene (IEG) Arc, which expresses in the nucleus following transcription before migrating to the cytoplasm over the next 25 min. Given this timeframe, we could identify whether Arc transcription was triggered by STFP recall, FCbP recall, or both. Contrary to past research, we found no differences in any Arc expression measures across a number of prefrontal regions and the ventral CA3 of the hippocampus between controls, demonstrators, and observers. We theorize that these results may indicate that relatively little Arc-dependent neural restructuring is taking place in the prefrontal cortices and ventral CA3 following recall of recently socially acquired information or directly acquired fear associations in these areas.

CO2 reactivity as a biomarker of exposure-based therapy non-response: study protocol.

BACKGROUND: Exposure-based therapy is an effective first-line treatment for anxiety-, obsessive-compulsive, and trauma- and stressor-related disorders; however, many patients do not improve, resulting in prolonged suffering and poorly used resources. Basic research on fear extinction may inform the development of a biomarker for the selection of exposure-based therapy. Growing evidence links orexin system activity to deficits in fear extinction and we have demonstrated that reactivity to an inhaled carbon dioxide (CO2) challenge-a safe, affordable, and easy-to-implement procedure-can serve as a proxy for orexin system activity and predicts fear extinction deficits in rodents. Building upon this basic research, the goal for the proposed study is to validate CO2 reactivity as a biomarker of exposure-based therapy non-response. METHODS: We will assess CO2 reactivity in 600 adults meeting criteria for one or more fear- or anxiety-related disorders prior to providing open exposure-based therapy. By incorporating CO2 reactivity into a multivariate model predicting treatment non-response that also includes reactivity to hyperventilation as well as a number of related predictor variables, we will establish the mechanistic specificity and the additive predictive utility of the potential CO2 reactivity biomarker. By developing models independently within two study sites (University of Texas at Austin and Boston University) and predicting the other site's data, we will validate that the results are likely to generalize to future clinical samples. DISCUSSION: Representing a necessary stage in translating basic research, this investigation addresses an important public health issue by testing an accessible clinical assessment strategy that may lead to a more effective treatment selection (personalized medicine) for patients with anxiety- and fear-related disorders, and enhanced understanding of the mechanisms governing exposure-based therapy. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT05467683 (20/07/2022).

The elegant complexity of fear in non-human animals.

Activation of the fear system is adaptive, and protects individuals from impending harm; yet, exacerbation of the fear system is at the source of anxiety-related disorders. Here, we briefly review the 'why' and 'how' of fear, with an emphasis on models that encapsulate the elegant complexity of rodents' behavioral responding in the face of impending harm, and its relevance to developing treatment interventions.

Friend recollections, and a collection of collaborations with Nadia.

In this selective review article, we showcase our collaborations with our colleague, Dr. Nadia Chaudhri. Dr. Chaudhri was an esteemed colleague and researcher who contributed greatly to our understanding of Pavlovian alcohol conditioning. From 2014 to 2019, we collaborated with Nadia. Here, we reflect on our friendship, the work we did together, and the continued impact on the field.

Hormonal contraceptives alter amphetamine place preference and responsivity in the intact female rat.

Hormonal contraceptives (HCs) containing synthetic ovarian hormones are commonly used among reproductive aged women; HCs alter the physiological state of the user by interfering with endogenous hormone concentrations and their actions on the reproductive tract. As ovarian hormones modulate the incidence of substance abuse disorders in women, this experiment explores how modulating female rat ovarian hormonal states with an HC containing the synthetic progestin levonorgestrel influences measures of drug preference and responsivity. First, rats underwent food-light Pavlovian conditioning to measure conditioned orienting, a known predictor of amphetamine (AMP) place preference. Then, rats were conditioned and tested for AMP place preference with either an HC implant or during estrous cycle stages associated with opposing ovarian hormone levels, that is, proestrus (P) or metestrus/diestrus (M/D), while recording ultrasonic vocalizations (USVs) as an index of he donic drug responsivity. Because of dopamine's (DA's) role in reward learning and memory, DA cell number and activity were examined using tyrosine hydroxylase and FOS immunohistochemistry after a final AMP challenge. Conditioned orienting did not differ between cycling and HC-implanted rats. HC rats emitted fewer USVs in response to AMP, showed marginally less AMP place preference, and had lower DA cell activity in the substantia nigra after AMP compared to P rats. M/D rats showed a similar behavioral profile and neural response as HC rats. This experiment suggests ovarian hormones affect drug preference and responsivity, while providing novel insight into how hormone-altering contraceptives may reduce these measures. (PsycInfo Database Record (c) 2022 APA, all rights reserved).

Sex differences in conditioned orienting and the role of estradiol in addiction-related behaviors.

Conditioned orienting response (OR) is a form of cue-directed behavior thought to indicate increased attentional and/or motivational processing of reward-associated stimuli. OR as a phenotype has been shown to predict both direct drug proclivity in female rats and behaviors indirectly related to drug proclivity in male rats, but no extant research has compared males and females in terms of their OR behavior or its notable substrates. As females are at increased risk for substance abuse, and the ovarian hormone estradiol is often cited as a driving factor for this predilection, it is important to characterize sex differences between males and females and explore what, if any, contribution estradiol has in behaviors which predict substance abuse. In these experiments, male and female rats [intact or ovariectomized (OVX) with/without estradiol replacement] were compared on a battery of behavioral tasks, including OR, novelty-seeking, attentional set-shifting, and ultrasonic vocalizations (USVs) to amphetamine treatment. Female rats, regardless of estradiol replacement, had higher OR scores than males. OR score was a predictor of attention impairments, and estradiol availability contributed to this relationship in females. Sex differences were not observed in novelty-seeking, attentional set-shifting, or USV response to amphetamine; however, estradiol replacement did alter the presentation of these behaviors. (PsycInfo Database Record (c) 2022 APA, all rights reserved).

Appetitive Behavior in the Social Transmission of Food Preference Paradigm Predicts Activation of Orexin-A producing Neurons in a Sex-Dependent Manner.

Orexin-producing cells in the lateral hypothalamic area have been shown to be involved in a wide variety of behavioral and cognitive functions, including the recall of appetitive associations and a variety of social behaviors. Here, we investigated the role of orexin in the acquisition and recall of socially transmitted food preferences in the rat. Rats were euthanized following either acquisition, short-term recall, or long-term recall of a socially transmitted food preference and their brains were processed for orexin-A and c-Fos expression. We found that while there were no significant differences in c-Fos expression between control and experimental subjects at any of the tested timepoints, females displayed significantly more activity in both orexinergic and non-orexinergic cells in the lateral hypothalamus. In the infralimbic cortex, we found that social behavior was significantly predictive of c-Fos expression, with social behaviors related to olfactory exploration appearing to be particularly influential. We additionally found that appetitive behavior was significantly predictive of orexin-A activity in a sex-dependent matter, with the total amount eaten correlating negatively with orexin-A/c-Fos colocalization in male rats but not female rats. These findings suggest a potential sex-specific role for the orexin system in balancing the stimulation of feeding behavior with the sleep/wake cycle.

Corrigendum: Gradual extinction prevents the return of fear: implications for the discovery of state.

[This corrects the article DOI: 10.3389/fnbeh.2013.00164.].

Altering Perceived Context: Transportation Cues Influence Novelty-Induced Context Exploration.

Context is the milieu in which everything occurs. Many research studies consider context, or even explicitly manipulate it; yet it remains challenging to characterize. We know that a context surrounds and influences tasks; however, the boundaries of its influence are difficult to define. In behavioral science, context is often operationalized by the physical environment in which the experiment takes place, and the boundaries of the context are assumed to begin at the entrance to that of the room or apparatus. Experiences during transportation to the testing space have been shown to impact rodent behavior and memory, but transportation's relationship with novelty and physical environment is not fully understood. The current study explored how familiar vs. novel cues, both within a physical environment and preceding it, impact the perception of a context. We manipulated context on three levels: physical testing environment, object cues within that environment, and transportation cues preceding entrance to the testing environment. We found that novel transportation cues can change rats' perception of both familiar and novel contexts. The effects of transportation on perceived context may be affected by the length of the retention interval, testing environment, and behavioral range. These data suggest that context is a broad concept that includes cues across time and is sensitive to small differences in experience.

Augmenting exposure therapy with pre-extinction fear memory reactivation and deepened extinction: A randomized controlled trial.

BACKGROUND: Pre-extinction fear memory reactivation (PE-FMR) and deepened extinction (DE) enhance long-term extinction of shock-conditioned fear, and may also enhance long-term extinction of naturally acquired fear. Preliminary data suggest that PE-FMR may additionally boost the speed of fear reduction during exposure therapy. DESIGN: Randomized controlled trial, factorial design. METHODS: Participants with elevated fears of either spiders or snakes were randomized to (1) exposure therapy alone (n = 41), (2) exposure therapy + PE-FMR (n = 42), (3) exposure therapy + DE (n = 41), or (4) exposure therapy + PE-FMR + DE (n = 42). Participants were assessed at baseline, post-treatment, and one-week follow-up on subjective and behavioral indices of phobia. Because treatment length was tailored to speed of fear reduction, survival analyses were used to examine the speed of fear reduction during treatment. RESULTS: DE did not improve clinical outcomes at post-treatment or follow-up, whereas PE-FMR produced more rapid fear reduction and was able to achive equivalent outcomes even when the duration of exposure therapy (tailored to speed of fear reduction) was shortened by an average of 21%. CONCLUSIONS: Data suggest that PE-FMR is a promising strategy for reducing the overall duration of exposure-based therapies. CLINICAL TRIAL REGISTRATION: (clinicaltrials.gov)NCT02160470.

Methylene Blue Preserves Cytochrome Oxidase Activity and Prevents Neurodegeneration and Memory Impairment in Rats With Chronic Cerebral Hypoperfusion.

Chronic cerebral hypoperfusion in neurocognitive disorders diminishes cytochrome oxidase activity leading to neurodegenerative effects and impairment of learning and memory. Methylene blue at low doses stimulates cytochrome oxidase activity and may thus counteract the adverse effects of cerebral hypoperfusion. However, the effects of methylene blue on cytochrome oxidase activity during chronic cerebral hypoperfusion have not been described before. To test this hypothesis, rats underwent bilateral carotid artery occlusion or sham surgery, received daily 4 mg/kg methylene blue or saline injections, and learned a visual water task. Brain mapping of cytochrome oxidase activity was done by quantitative enzyme histochemistry. Permanent carotid occlusion for 1 month resulted in decreased cytochrome oxidase activity in visual cortex, prefrontal cortex, perirhinal cortex, hippocampus and amygdala, and weaker interregional correlation of cytochrome oxidase activity between these regions. Methylene blue preserved cytochrome oxidase activity in regions affected by carotid occlusion and strengthened their interregional correlations of cytochrome oxidase activity, which prevented neurodegenerative effects and facilitated task-specific learning and memory. Brain-behavior correlations revealed positive correlations between performance and brain regions in which cytochrome oxidase activity was preserved by methylene blue. These results are the first to demonstrate that methylene blue prevents neurodegeneration and memory impairment by preserving cytochrome oxidase activity and interregional correlation of cytochrome oxidase activity in brain regions susceptible to chronic hypoperfusion. This demonstration provides further support for the hypothesis that lower cerebral blood flow results in an Alzheimer's-like syndrome and that stimulating cytochrome oxidase activity with low-dose methylene blue is neuroprotective.

Mapping the estrous cycle to context-specific extinction memory.

In Pavlovian renewal paradigms, intact female rats have previously failed to exhibit renewal of appetitive behavior after extinction. However, when treated with exogenous estradiol, female rats exhibit robust renewal behavior. The current study aims to investigate whether the estrous cycle can influence renewal of appetitive behaviors and activity in brain areas known to support the renewal effect. We further aimed to examine whether the estrous cycle would similarly affect renewal of two different types of appetitive behaviors. We first establish that rats in the proestrous stage of the estrous cycle during extinction exhibit elevated renewal behavior compared with rats in either metestrous/diestrous stages, and only rats in proestrus during extinction training (but not during the renewal test) exhibit elevated renewal behavior. Furthermore, we show that this estrous cycle dependent effect on renewal only applies to the conditioned approach behavior toward the food delivery site but not the conditioned approach behavior toward the light cue associated with food delivery. Finally, we examined FOS activity within the prelimbic and infralimbic areas of the medial prefrontal cortex, the dorsal and ventral hippocampal formation, the paraventricular nucleus of the thalamus, the nucleus accumbens, and areas of the amygdala. Particularly in the hippocampus and amygdala, FOS expression which corresponded to the behavioral differences between groups was observed. Results from this study suggest that context information processing may vary as a function of endogenous female hormones across the gonadal hormone cycle and that encoding and retrieval of this information is accomplished in a state-specific manner. (PsycINFO Database Record (c) 2019 APA, all rights reserved).