A mild stressor induces short-term anxiety and long-term phenotypic changes in trauma-related behavior in female rats.

Introduction: Anxiety and anxiety-influenced disorders are sexually dimorphic with women being disproportionately affected compared to men. Given the increased prevalence in women and the documented differences in anxiety and trauma behavior between male and female rats this paper sought to examine the link between stress, anxiety, and fear learning and extinction in female rats. We tested the hypothesis that a mild stressor will induce short-and long-term increases in anxiety and produce long term effects on subsequent fear learning and extinction behavior. Methods: We induced anxiety in female Sprague- Dawley rats with a short (3 min) exposure to a ball of cat hair infused with 150 µl of cat urine (mild stressor) that elicits innate fear but does not cause fear conditioning. The control group was exposed to fake cat hair. Anxiety was assessed in the Light-Dark Open Field (LDOF) or Elevated Plus Maze (EPM) before, immediately after and 4 days after stimulus exposure. Two weeks later, all animals were subject to Contextual Fear Conditioning (CFC) in the Shock Arm of a Y-maze, blocked off from the rest of the maze. Memory and fear extinction (learning of safety) was assessed in the following four days by placing each rat in one of the Safe Arms and measuring avoidance extinction (time spent and number of entries in the Shock Arm). Results: Cat hair exposure induced changes in anxiety-like behavior in the short-term that appeared resolved 4 days later. However, the cat-hair exposed rats had long-term (2 weeks) phenotypic changes expressed as altered exploratory behavior in an emotionally neutral novel place. Fear learning and extinction were not impaired. Yet, using avoidance extinction, we demonstrated that the phenotypic difference induced by the mild stressor could be documented and dissociated from learning and memory. Discussion: These findings demonstrate that the history of stress, even mild stress, has subtle long-term effects on behavior even when short-term anxiety appears resolved.

Neuroinflammation is a susceptibility factor in developing a PTSD-like phenotype.

Introduction: Post-Traumatic Stress Disorder (PTSD) is a psychological disorder that occurs after a traumatic event in a subset of exposed individuals. This implies the existence of susceptibility factors that foster the development of PTSD. Susceptibility factors are present before trauma and can contribute to the development and maintenance of PTSD after trauma. Manipulation of susceptibility factors may decrease the probability of developing PTSD. A putative susceptibility factor is inflammation. Patients with PTSD have been documented to have a higher pro-inflammatory profile compared to non-PTSD subjects. In addition, they are more likely to develop and die from cardiovascular disease which has a strong inflammation component. It is not known, however, whether inflammation plays a role in developing PTSD or whether reducing inflammation can prevent PTSD. Methods: We used the Revealing Individual Susceptibility to a PTSD-like phenotype (RISP) model to behaviorally classify male rats as resilient or susceptible before trauma and tested their serum and prefrontal cortical (mPFC) levels of IL-1ß, IL-6, TNFα, IL-10, IFN IFNγ, and KC/GRO to determine whether inflammation represents a putative susceptibility factor for PTSD. Results: We found elevated IL-6 levels in the mPFC, but not serum, of susceptible rats compared to resilient animals before trauma. Serum and mPFC levels were not correlated in any of the cytokines/chemokines. Rats with high anxiety-like behavior had elevated IL-6 and IL-10 mPFC levels. Acoustic startle responses were not associated with cytokine/chemokine levels. Discussion: Neuroinflammation, rather than systemic inflammation exists in susceptible male rats before trauma and is thus a putative susceptibility factor for PTSD. Thus, susceptibility appears neurogenic in its pathogenesis. The lack of differences between susceptible and resilient rats in serum cytokine/chemokine levels infers that peripheral markers will not be useful in determining susceptibility. Chronic neuroinflammation appears more broadly associated with anxiety rather than startle responses.

Sex Differences In Avoidance Extinction After Contextual Fear Conditioning: Anxioescapic Behavior In Female Rats.

Fear memories are important for survival and are implicated in the etiology of fear disorders such as Post Traumatic Stress Disorder (PTSD). Fear memories are well studied pre-clinically and sex differences in rodent fear expression have been reported: females tend to freeze less than males. Whether this is a difference in fear learning or expression is debated. We aimed to differentiate between these possibilities with a task that allowed female rats to express fear memory by moving, rather than freezing. We assessed fear extinction after contextual fear conditioning in the isolated Shock Arm of a Y-maze in female and male rats by either placing them back in the isolated Shock Arm (Fear Extinction in the Shock Context) or allowing them to move freely in the Y-maze during extinction training and enter/avoid the Shock Arm (Avoidance Extinction). We confirmed that female rats freeze less than males during fear extinction in both settings. During Avoidance Extinction, however, both sexes had similar avoidance of the Shock Context, showing comparable fear memory and extinction. Additionally, female rats made more entries into the non-shock arms. Thus, female and male rats have similar fear learning but females express it with an active motor response. Furthermore, female rats also exhibited an active motor response under other anxiogenic conditions (Elevated Plus Maze) and had higher reactivity (Acoustic Startle Response) but not when fear-eliciting stimuli were present: cat hair and foot-shock. In summary, female rats have an active motor response to anxiogenic stimuli which we termed 'Anxioescapic' behavior strategy.

Emotional state alters encoding of long-term spatial episodic memory.

The neurobiology of emotion and episodic memory are well-researched subjects, as is their intersection: memory of emotional events (i.e. emotional memory). We and others have previously demonstrated that the emotional valence of stimuli is encoded in the dorsal hippocampus, a structure integral to the acquisition, consolidation and retrieval of long-term episodic memories. Such findings are consistent with the idea that the emotional valence of stimuli contributes to the "what" component of episodic memories ("where" and "when" being the other components). We hypothesized that being in a heightened emotional state by itself does not contribute to the "what" component of episodic memories. We tested an inference of this hypothesis - that negative emotional state does not alter re-encoding of a spatial episodic event. Rats from the experimental group explored a novel place at their baseline emotional state (Event 1) and 20 min later re-explored the same place (Event 2) in a negative emotional state induced by a state-altering event prior to Event 2. We examined neuronal ensembles that induced expression of Arc and Homer1a, two immediate-early genes (IEGs) necessary for synaptic plasticity and consolidation of long-term memories, during both events. We found that in dorsal CA1 and dorsal CA3, Event 1 and Event 2 induced IEG expression in different neuronal ensembles. This finding was reflected in a low Fidelity score, which assesses the percentage of the Event 1 IEG-expressing ensemble re-activated during Event 2. The Fidelity score was significantly higher in a control group which was at a baseline emotional state during Event 2. Groups which were matched for non-specific disruptions from the state-altering event had intermediate Fidelity scores in dorsal CA1. The Fidelity scores of the dorsal CA3 in the latter groups were similar to those of the control group. Combined, the findings reject the tested hypothesis and suggest that a negative emotional state is encoded in the hippocampus as part of the long-term memory of episodic events that lack explicit emotion-inducing stimuli. These findings also suggest that individuals who often experience strong negative emotional states incorporate these states into ongoing non-emotional episodic memories.

Light-Dark Open Field (LDOF): A novel task for sensitive assessment of anxiety.

BACKGROUND: Pre-clinical studies of psychiatric disorders often include a measure of anxiety-like behavior. Several tasks exist that serve this purpose, but because anxiety is complex with a myriad of anxiogenic stimuli, researchers are often compelled to use multiple tasks. The Light-Dark Open Field (LDOF) combines concepts from two such tasks, Light-Dark Box and Open Field, into one task with the synergistic effect of enhanced discrimination of anxiety-like behavior. NEW METHODS: Our goal was to increase the sensitivity of the Open Field task with the addition of a shadow, conceptually similar to the Light-Dark Box, to detect concealed differences even under bright light, which is highly anxiogenic. The resulting LDOF allows assessment of anxiety due to bright light and open space simultaneously, while retaining the ability to assess the impact of each with custom indices. In addition, it maintains all the advantages and measures of the Open Field. RESULTS: Using custom created indices from measures collected in the LDOF one can assess anxiety induced by light, open space, or light and open space combined and thus elucidate anxiety-inducing factors. Using two strains of rats: an outbred strain, Sprague-Dawley (SD), and a strain that exhibits high trait anxiety, Lewis rats, we found that increased discrimination for anxiety-like behavior can be achieved with the Light-Dark Open Field. COMPARISON WITH EXISTING MODELS: The LDOF allows researchers to extract the traditional measures of an Open Field, including valuable information about locomotion and habituation while adding a further layer of discrimination with the light-dark component. Because the LDOF is a combination of two different tests, it saves time compared to running multiple experiments in series that then need to be counterbalanced to reduce artefacts and task order effects. In addition, it detects differences even when traditional tasks of anxiety have reached their ceiling sensitivity (i.e. EPM under bright light conditions). CONCLUSION: We present the Light-Dark Open Field: a simple modification of an existing Open Field apparatus that incorporates aspects of the Light-Dark Box with the addition of a shadow. The shadow (Dark Perimeter) allows for increased discrimination in detecting anxiety-like behaviors. Comparison of anxiety-like behavior between Lewis and SD rats allowed us to develop the construct and face validity of the LDOF as well as demonstrate its sensitivity even under bright light conditions. In addition, we developed 3 indices that allow one to parse out, from one set of data, the effect of two anxiogenic stimuli- bright light and open space.

Investigating Individual Pre-trauma Susceptibility to a PTSD-Like Phenotype in Animals.

Post-Traumatic Stress Disorder (PTSD) is a complex condition that develops after experiencing a severe emotional trauma, with or without physical trauma. There is no known cure and evidence-based treatments, which are effective in reducing symptoms, have low retention rates. It is therefore important, in addition to seeking new therapeutics, to identify ways to reduce the likelihood of developing PTSD. The fact that some, but not all, individuals exposed to the same traumatic event develop PTSD suggests that there is individual susceptibility. Investigating susceptibility and underlying factors will be better guided if there is a coherent framework for such investigations. In this review, we propose that susceptibility is a dynamic state that is comprised of susceptibility factors (before trauma) and sequalae factors (during or after trauma, but before PTSD diagnosis). We define key features of susceptibility and sequalae factors as: (1) they are detectable before trauma (susceptibility factors) or during/shortly after trauma (sequalae factors), (2) they can be manipulated, and (3) manipulation of these factors alters the likelihood of developing PTSD, thus affecting resilience. In this review we stress the importance of investigating susceptibility to PTSD with appropriate animal models, because prospective human studies are expensive and manipulation of susceptibility and sequalae factors for study purposes may not always be feasible. This review also provides a brief overview of a subset of animal models that study PTSD-related behaviors and related alterations in endocrine and brain systems that focus on individual differences, peri- and post-trauma. Attention is drawn to the RISP model (Revealing Individual Susceptibility to a PTSD-like Phenotype) which assesses susceptibility before trauma. Using the RISP model and expression of plasticity-associated immediate early genes, Arc and Homer1a, we have identified impaired hippocampal function as a potential susceptibility factor. We further discuss other putative susceptibility factors and approaches to mitigate them. We assert that this knowledge will guide successful strategies for interventions before, during or shortly after trauma that can decrease the probability of developing PTSD.

Sex-dependent effects of early life inflammatory pain on sucrose intake and sucrose-associated hippocampal Arc expression in adult rats.

We hypothesize that dorsal hippocampal (dHC) neurons, which are critical for episodic memory, form a memory of a meal and inhibit the initiation of the next meal and the amount ingested during that meal. In support, we showed previously that (1) consuming a sucrose meal induces expression of the synaptic plasticity marker activity-regulated cytoskeleton-associated protein (Arc) in dHC neurons and (2) reversible inactivation of these neurons immediately following a sucrose meal accelerates the onset of the next meal and increases the size of that meal. These data suggest that hippocampal-dependent memory inhibits intake; therefore, the following experiments were conducted to determine whether hippocampal-dependent memory impairments are associated with increased intake. We reported recently that one episode of early life inflammatory pain impairs dHC-dependent memory in adult rats. The present study determined whether neonatal inflammatory pain also increases sucrose intake and attenuates sucrose-associated Arc expression. Male and female Sprague-Dawley rats were given an intraplantar injection of the inflammatory agent carrageenan (1%) on the day of birth and sucrose intake and sucrose-associated dHC Arc expression were measured in adulthood. Neonatal inflammatory pain increased sucrose intake in adult female and male rats, decreased sucrose-associated dHC Arc expression in female rats, and tended to have a similar effect on Arc expression in male rats. Neonatal inflammatory pain significantly decreased the interval between two sucrose meals in female but not in male rats. Morphine administration at the time of insult attenuated the effects of injury on sucrose intake. Collectively, these findings indicate that one brief episode of inflammatory pain on the day of birth has a long long-lasting, sex-dependent impact on intake of a palatable food in adulthood.

Ventral hippocampal neurons inhibit postprandial energy intake.

Evidence suggests that the memory of a recently ingested meal limits subsequent intake. Given that ventral hippocampal (vHC) neurons are involved in memory and energy intake, the present experiment tested the hypothesis that vHC neurons contribute to the formation of a memory of a meal and inhibit energy intake during the postprandial period. We tested (1) whether pharmacological inactivation of vHC neurons during the period following a sucrose meal, when the memory of the meal would be undergoing consolidation, accelerates the onset of the next sucrose meal and increases intake and (2) whether sucrose intake increases vHC expression of the synaptic plasticity marker activity-regulated cytoskeletal-associated protein (Arc). Adult male Sprague-Dawley rats were trained to consume a 32% sucrose solution daily at the same time and location. On the experimental day, the rats were given intra-vHC infusions of the GABAA receptor agonist muscimol or vehicle after they finished their first sucrose meal. Compared to vehicle infusions, postmeal intra-vHC muscimol infusions decreased the latency to the next sucrose meal, increased the amount of sucrose consumed during that meal, increased the total number of sucrose meals and the total amount of sucrose ingested. In addition, rats that consumed sucrose had higher levels of Arc expression in both vHC CA1 and CA3 subfields than cage control rats. Collectively, these findings are the first to show that vHC neurons inhibit energy intake during the postprandial period and support the hypothesis that vHC neurons form a memory of a meal and inhibit subsequent intake. © 2016 Wiley Periodicals, Inc.

Sweet orosensation induces Arc expression in dorsal hippocampal CA1 neurons in an experience-dependent manner.

There is limited knowledge regarding how the brain controls the timing of meals. Similarly, there is a large gap in our understanding of how top-down cognitive processes, such as memory influence energy intake. We hypothesize that dorsal hippocampal (dHC) neurons, which are critical for episodic memory, form a memory of a meal and inhibit meal onset during the postprandial period. In support, we showed previously that reversible inactivation of these neurons during the period following a sucrose meal accelerates the onset of the next meal. If dHC neurons form a memory of a meal, then consumption should induce synaptic plasticity in dHC neurons. To test this, we determined (1) whether a sucrose meal increases the expression of the synaptic plasticity marker activity-regulated cytoskeleton-associated protein (Arc) in dHC CA1 neurons, (2) whether previous experience with sucrose influences sucrose-induced Arc expression, and (3) whether the orosensory stimulation produced by the noncaloric sweetener saccharin is sufficient to induce Arc expression. Male Sprague-Dawley rats were trained to consume a sweetened solution at a scheduled time daily. On the experimental day, they were given a solution for 7 min, euthanized, and then fluorescence in situ hybridization procedures were used to measure meal-induced Arc mRNA. Compared to caged control rats, Arc expression was significantly higher in rats that consumed sucrose or saccharin. Interestingly, rats given additional experience with sucrose had less Arc expression than rats with less sucrose experience, even though both groups consumed similar amounts on the experimental day. Thus, this study is the first to suggest that orosensory stimulation produced by consuming a sweetened solution and possibly the hedonic value of that sweet stimulation induces synaptic plasticity in dHC CA1 neurons in an experience-dependent manner. Collectively, these findings are consistent with our hypothesis that dHC neurons form a memory of a meal.

Influence of Isoflurane on Immediate-Early Gene Expression.

BACKGROUND: Anterograde amnesia is a hallmark effect of volatile anesthetics. Isoflurane is known to affect both the translation and transcription of plasticity-associated genes required for normal memory formation in many brain regions. What is not known is whether isoflurane anesthesia prevents the initiation of transcription or whether it halts transcription already in progress. We tested the hypothesis that general anesthesia with isoflurane prevents learning-induced initiation of transcription of several memory-associated immediate-early genes (IEGs) correlated with amnesia; we also assessed whether it stops transcription initiated prior to anesthetic administration. METHODS: Using a Tone Fear Conditioning paradigm, rats were trained to associate a tone with foot-shock. Animals received either no anesthesia, anesthesia immediately after training, or anesthesia before, during, and after training. Animals were either sacrificed after training or tested 24 h later for long-term memory. Using Cellular Compartment Analysis of Temporal Activity by Fluorescence in situ Hybridization (catFISH), we examined the percentage of neurons expressing the IEGs Arc/Arg3.1 and Zif268/Egr1/Ngfi-A/Krox-24 in the dorsal hippocampus, primary somatosensory cortex, and primary auditory cortex. RESULTS: On a cellular level, isoflurane administered at high doses (general anesthesia) prevented initiation of transcription, but did not stop transcription of Arc and Zif268 mRNA initiated prior to anesthesia. On a behavioral level, the same level of isoflurane anesthesia produced anterograde amnesia for fear conditioning when administered before and during training, but did not produce retrograde amnesia when administered immediately after training. CONCLUSION: General anesthesia with isoflurane prevents initiation of learning-related transcription but does not stop ongoing transcription of two plasticity-related IEGs, Arc and Zif268, a pattern of disruption that parallels the effects of isoflurane on memory formation. Combined with published research on the effects of volatile anesthetics on memory in behaving animals, our data suggests that different levels of anesthesia affect memory via different mechanisms: general anesthesia prevents elevation of mRNA levels of Arc and Zif268 which are necessary for normal memory formation, while anesthesia at lower doses affects the strength of memory by affecting levels of plasticity-related proteins.

Altered hippocampal function before emotional trauma in rats susceptible to PTSD-like behaviors.

Posttraumatic stress disorder (PTSD) is an anxiety disorder that occurs after experiencing a traumatic event. Susceptibility to PTSD exists, as only some trauma-exposed individuals develop this condition. Investigating susceptibilities in animal models can contribute to understanding the etiology of the disorder. We previously reported an animal model which allows reliable pre-classification of rats as susceptible (Sus) or resistant (Res) to developing a PTSD-like phenotype after a later trauma. Here we report that Sus, compared to Res, rats have altered hippocampal function, along the septo-temporal axis, prior to experiencing a traumatic event. In Experiment I, Res and Sus rats explored a novel box twice. Using a cellular imaging method for assessing plasticity-related immediate-early gene expression in large neuronal ensembles, Arc/Homer1a catFISH, we show that Sus rats have smaller vCA3 ensembles during the second exploration. This suppressed vCA3 activation in Sus rats was not due to a difference in exploratory behavior, or to a difference in Arc/Homer1a expression in the basolateral amygdala (BLA). BLA is a main source of inputs to vCA3, but both the ensemble size and overlap of BLA ensembles activated during the two explorations was similar to that of Res rats. Additionally, Sus rats had significant 'infidelity' in their dorsal hippocampal representations of the second event: a lower overlap, compared to Res rats, of Arc/Homer1a-expressing ensembles activated during the two explorations (the size of the ensembles were similar to those of Res rats). These differences were revealed only in conditions of relatively low stress, because they were not observed when Sus and Res rats experienced fear conditioning (Experiment II). Combined, the findings show that altered hippocampal function exists before experiencing emotional trauma in susceptible rats and suggest that this is a risk factor for PTSD.

Encoding of emotion-paired spatial stimuli in the rodent hippocampus.

Rats can acquire the cognitive component of CS-US associations between sensory and aversive stimuli without a functional basolateral amygdala (BLA). Thus, other brain regions should support such associations. Some septal/dorsal CA1 (dCA1) neurons respond to both spatial stimuli and footshock, suggesting that dCA1 could be one such region. We report that, in both dorsal and ventral hippocampus, different neuronal ensembles express immediate-early genes (IEGs) when a place is experienced alone vs. when it is associated with foot shock. We assessed changes in the size and overlap of hippocampal neuronal ensembles activated by two behavioral events using a cellular imaging method, Arc/Homer1a catFISH. The control group (A-A) experienced the same place twice, while the experimental group (A-CFC) received the same training plus two foot shocks during the second event. During fear conditioning, A-CFC, compared to A-A, rats had a smaller ensemble size in dCA3, dCA1, and vCA3, but not vCA1. Additionally, A-CFC rats had a lower overlap score in dCA1 and vCA3. Locomotion did not correlate with ensemble size. Importantly, foot shocks delivered in a training paradigm that prevents establishing shock-context associations, did not induce significant Arc expression, rejecting the possibility that the observed changes in ensemble size and composition simply reflect experiencing a foot shock. Combined with data that Arc is necessary for lasting synaptic plasticity and long-term memory, the data suggests that Arc/H1a+ hippocampal neuronal ensembles encode aspects of fear conditioning beyond space and time. Rats, like humans, may use the hippocampus to create integrated episodic-like memory during fear conditioning.

Predicting impaired extinction of traumatic memory and elevated startle.

BACKGROUND: Emotionally traumatic experiences can lead to debilitating anxiety disorders, such as phobias and Post-Traumatic Stress Disorder (PTSD). Exposure to such experiences, however, is not sufficient to induce pathology, as only up to one quarter of people exposed to such events develop PTSD. These statistics, combined with findings that smaller hippocampal size prior to the trauma is associated with higher risk of developing PTSD, suggest that there are pre-disposing factors for such pathology. Because prospective studies in humans are limited and costly, investigating such pre-dispositions, and thus advancing understanding of the genesis of such pathologies, requires the use of animal models where predispositions are identified before the emotional trauma. Most existing animal models are retrospective: they classify subjects as those with or without a PTSD-like phenotype long after experiencing a traumatic event. Attempts to create prospective animal models have been largely unsuccessful. METHODOLOGY/PRINCIPAL FINDINGS: Here we report that individual predispositions to a PTSD-like phenotype, consisting of impaired rate and magnitude of extinction of an emotionally traumatic event coupled with long-lasting elevation of acoustic startle responses, can be revealed following exposure to a mild stressor, but before experiencing emotional trauma. We compare, in rats, the utility of several classification criteria and report that a combination of criteria based on acoustic startle responses and behavior in an anxiogenic environment is a reliable predictor of a PTSD-like phenotype. CONCLUSIONS/SIGNIFICANCE: There are individual predispositions to developing impaired extinction and elevated acoustic startle that can be identified after exposure to a mildly stressful event, which by itself does not induce such a behavioral phenotype. The model presented here is a valuable tool for studying the etiology and pathophysiology of anxiety disorders and provides a platform for testing behavioral and pharmacological interventions that can reduce the probability of developing pathologic behaviors associated with such disorders.