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.

Angiotensin receptor (AT2R) agonist C21 prevents cognitive decline after permanent stroke in aged animals-A randomized double- blind pre-clinical study.

Post stroke cognitive impairment (PSCI) is an understudied, long-term complication of stroke, impacting nearly 30-40% of all stroke survivors. No cure is available once the cognitive deterioration manifests. To our knowledge, this is the first study to investigate the long-term effects of C21 treatment on the development of PSCI in aged animals. Treatments with C21 or vehicle were administered orally, 24 h post-stroke, and continued for 30 days. Outcome measures for sensorimotor and cognitive function were performed using a sequence of tests, all blindly conducted and assessed at baseline as well as at different time points post-stroke. Our findings demonstrate that the angiotensin receptor (AT2R) agonist C21 effectively prevents the development of PSCI in aged animals.

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.

Long noncoding RNA NEAT1 (nuclear paraspeckle assembly transcript 1) is critical for phenotypic switching of vascular smooth muscle cells.

In response to vascular injury, vascular smooth muscle cells (VSMCs) may switch from a contractile to a proliferative phenotype thereby contributing to neointima formation. Previous studies showed that the long noncoding RNA (lncRNA) NEAT1 is critical for paraspeckle formation and tumorigenesis by promoting cell proliferation and migration. However, the role of NEAT1 in VSMC phenotypic modulation is unknown. Herein we showed that NEAT1 expression was induced in VSMCs during phenotypic switching in vivo and in vitro. Silencing NEAT1 in VSMCs resulted in enhanced expression of SM-specific genes while attenuating VSMC proliferation and migration. Conversely, overexpression of NEAT1 in VSMCs had opposite effects. These in vitro findings were further supported by in vivo studies in which NEAT1 knockout mice exhibited significantly decreased neointima formation following vascular injury, due to attenuated VSMC proliferation. Mechanistic studies demonstrated that NEAT1 sequesters the key chromatin modifier WDR5 (WD Repeat Domain 5) from SM-specific gene loci, thereby initiating an epigenetic "off" state, resulting in down-regulation of SM-specific gene expression. Taken together, we demonstrated an unexpected role of the lncRNA NEAT1 in regulating phenotypic switching by repressing SM-contractile gene expression through an epigenetic regulatory mechanism. Our data suggest that NEAT1 is a therapeutic target for treating occlusive vascular diseases.

Role of angiotensin system modulation on progression of cognitive impairment and brain MRI changes in aged hypertensive animals - A randomized double- blind pre-clinical study.

Growing evidence suggests that renin angiotensin system (RAS) modulators support cognitive function in various animal models. However, little is known about their long-term effects on the brain structure in aged hypertensive animals with chronic cerebral hypoperfusion as well as which specific domains of cognition are most affected. Therefore, in the current study we examined the effects of Candesartan and Compound 21 (C21) (RAS modulators) on aspects of cognition known to diminish with advanced age and accelerate with hypertension and vascular disease. Outcome measures for sensorimotor and cognitive function were performed using a sequence of tests, all blindly conducted and assessed at baseline and after 4 and 8 weeks of chronic hypoxic hypoperfusion and treatment. Magnetic resonance imaging (MRI) was performed at the end of the 8 week study period followed by animal sacrifice and tissue collection. Both Candesartan and C21 effectively preserved cognitive function and prevented progression of vascular cognitive impairment (VCI) but only candesartan prevented loss of brain volume in aged hypertensive animals. Collectively, our findings demonstrate that delayed administration of RAS modulators effectively preserve cognitive function and prevent the development / progression of VCI in aged hypertensive animals with chronic cerebral hypoperfusion.

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.