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.

CARMN Is an Evolutionarily Conserved Smooth Muscle Cell-Specific LncRNA That Maintains Contractile Phenotype by Binding Myocardin.

BACKGROUND: Vascular homeostasis is maintained by the differentiated phenotype of vascular smooth muscle cells (VSMCs). The landscape of protein coding genes comprising the transcriptome of differentiated VSMCs has been intensively investigated but many gaps remain including the emerging roles of noncoding genes. METHODS: We reanalyzed large-scale, publicly available bulk and single-cell RNA sequencing datasets from multiple tissues and cell types to identify VSMC-enriched long noncoding RNAs. The in vivo expression pattern of a novel smooth muscle cell (SMC)-expressed long noncoding RNA, Carmn (cardiac mesoderm enhancer-associated noncoding RNA), was investigated using a novel Carmn green fluorescent protein knock-in reporter mouse model. Bioinformatics and quantitative real-time polymerase chain reaction analysis were used to assess CARMN expression changes during VSMC phenotypic modulation in human and murine vascular disease models. In vitro, functional assays were performed by knocking down CARMN with antisense oligonucleotides and overexpressing Carmn by adenovirus in human coronary artery SMCs. Carotid artery injury was performed in SMC-specific Carmn knockout mice to assess neointima formation and the therapeutic potential of reversing CARMN loss was tested in a rat carotid artery balloon injury model. The molecular mechanisms underlying CARMN function were investigated using RNA pull-down, RNA immunoprecipitation, and luciferase reporter assays. RESULTS: We identified CARMN, which was initially annotated as the host gene of the MIR143/145 cluster and recently reported to play a role in cardiac differentiation, as a highly abundant and conserved, SMC-specific long noncoding RNA. Analysis of the Carmn GFP knock-in mouse model confirmed that Carmn is transiently expressed in embryonic cardiomyocytes and thereafter becomes restricted to SMCs. We also found that Carmn is transcribed independently of Mir143/145. CARMN expression is dramatically decreased by vascular disease in humans and murine models and regulates the contractile phenotype of VSMCs in vitro. In vivo, SMC-specific deletion of Carmn significantly exacerbated, whereas overexpression of Carmn markedly attenuated, injury-induced neointima formation in mouse and rat, respectively. Mechanistically, we found that Carmn physically binds to the key transcriptional cofactor myocardin, facilitating its activity and thereby maintaining the contractile phenotype of VSMCs. CONCLUSIONS: CARMN is an evolutionarily conserved SMC-specific long noncoding RNA with a previously unappreciated role in maintaining the contractile phenotype of VSMCs and is the first noncoding RNA discovered to interact with myocardin.

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.