Single-Nucleus RNA-Sequencing Profiling of Mouse Lung. Reduced Dissociation Bias and Improved Rare Cell-Type Detection Compared with Single-Cell RNA Sequencing.

Single-cell RNA sequencing (scRNASeq) has advanced our understanding of lung biology, but its utility is limited by the need for fresh samples, loss of cell types by death or inadequate dissociation, and transcriptional stress responses induced during tissue digestion. Single-nucleus RNA sequencing (snRNASeq) has addressed these deficiencies in other tissues, but no protocol exists for lung tissue. We present a snRNASeq protocol and compare its results with those of scRNASeq. Two nuclear suspensions were prepared in lysis buffer on ice while one cell suspension was generated using enzymatic and mechanical dissociation. Cells and nuclei were processed using the 10× Genomics platform, and sequencing data were analyzed by Seurat. A total of 16,110 single-nucleus and 11,934 single-cell transcriptomes were generated. Gene detection rates were equivalent in snRNASeq and scRNASeq (∼1,700 genes and 3,000 unique molecular identifiers per cell) when mapping intronic and exonic reads. In the combined data, 89% of epithelial cells were identified by snRNASeq versus 22.2% of immune cells. snRNASeq transcriptomes are enriched for transcription factors and signaling proteins, with reduction in mitochondrial and stress-response genes. Both techniques improved mesenchymal cell detection over previous studies. Homeostatic signaling relationships among alveolar cell types were defined by receptor-ligand mapping using snRNASeq data, revealing interplay among epithelial, mesenchymal, and capillary endothelial cells. snRNASeq can be applied to archival murine lung samples, improves dissociation bias, eliminates artifactual gene expression, and provides similar gene detection compared with scRNASeq.

Cooperatively enhanced reactivity and "stabilitaxis" of dissociating oligomeric proteins.

Many functional units in biology, such as enzymes or molecular motors, are composed of several subunits that can reversibly assemble and disassemble. This includes oligomeric proteins composed of several smaller monomers, as well as protein complexes assembled from a few proteins. By studying the generic spatial transport properties of such proteins, we investigate here whether their ability to reversibly associate and dissociate may confer on them a functional advantage with respect to nondissociating proteins. In uniform environments with position-independent association-dissociation, we find that enhanced diffusion in the monomeric state coupled to reassociation into the functional oligomeric form leads to enhanced reactivity with localized targets. In nonuniform environments with position-dependent association-dissociation, caused by, for example, spatial gradients of an inhibiting chemical, we find that dissociating proteins generically tend to accumulate in regions where they are most stable, a process that we term "stabilitaxis."

Effects of 35% carbon dioxide (CO2) inhalation in patients with post-traumatic stress disorder (PTSD): A double-blind, randomized, placebo-controlled, cross-over trial.

BACKGROUND: In patients with post-traumatic stress disorder (PTSD) two open pilot studies about the effects of 35% carbon dioxide (CO2) exist. One shows an augmented panicogenic and anxiogenic response (Muhtz et al., 2011), the other does not (Talesnik et al. 2007). We further characterized the CO2 reactivity in PTSD using for the first time placebo-controlled and double-blind conditions. METHODS: In 20 patients with PTSD we assessed panic, anxiety, dissociative and PTSD symptoms after a single vital capacity inhalation of 35% CO2 compared to a placebo gas condition in a within-participant cross-over, placebo-controlled, double-blind and randomized design. RESULTS: Inhalation of 35% CO2 versus placebo provoked significantly increased panic, anxiety, dissociative and PTSD symptoms. The reaction to placebo gas was minimal. Order of inhalation, patients' sex or age did not influence the results. The panic and anxiety response under CO2 was considerably higher in the PTSD patients than in healthy controls from our previous open study. CONCLUSIONS: The results corroborate that our preceding findings of an increased CO2 reactivity in patients with PTSD are not false positive due to the open design or the lack of placebo control. Replication in a larger number of PTSD patients and matched control subjects is needed. The potential role of childhood traumatisation, psychiatric comorbidity, psychotropic medication and trait dissociation in prior contradictory reports should be clarified.

NMDA receptor antagonists for depression: Critical considerations.

BACKGROUND: Research studies suggest that glutamate dysfunction, in particular N-methyl-D-aspartate receptors (NMDARs) abnormalities, may be involved in the pathophysiology of major neuropsychiatric conditions. Increased glutamatergic excitotoxic activity may be found in some brain circuits of patients with major depression. According to several published reports, NMDAR antagonists may exert antidepressant activity, but the molecular changes associated with abnormal glutamatergic neurotransmission remain unclear. METHODS: We have critically reviewed the current literature in order to investigate the role of NMDAR antagonists in major depression. RESULTS: NMDAR antagonists, such as ketamine, may be considered novel and promising pharmacological options for the rapid treatment of treatment-resistant depression patients. This is in contrast to the delayed action of the currently available antidepressant medications. Studies suggest that glutamatergic receptor modulation may enhance neuroplasticity mechanisms and neurogenesis together with the release of some neurotransmitters. Unfortunately, the use of ketamine is currently limited by some transient adverse events, including dissociative symptoms. CONCLUSIONS: Targeting NMDARs using antagonists represents an important alternative antidepressant option in major depression. However, NMDAR antagonists may exert different actions based on the differential brain location of NMDAR.

Dissociation in borderline personality disorder: Disturbed cognitive and emotional inhibition and its neural correlates.

Evidence is heterogeneous regarding whether patients with borderline personality disorder (BPD) display disturbed emotional inhibition in the emotional Stroop task. Previous findings suggest that state dissociation may influence cognitive inhibition of task-irrelevant material, particularly with negative content. Our aim was to examine performance in an emotional Stroop task including negative, neutral, and positive words in BPD patients and healthy controls during functional magnetic resonance imaging. In advance, half of the BPD patients underwent a dissociation induction using script-driven imagery. BPD patients without dissociation induction showed behavioural performance comparable to that of healthy controls but displayed stronger neural responses, especially to positive stimuli, in the superior temporal gyrus, dorsomedial prefrontal cortex, and anterior cingulate cortex. BPD patients with dissociation induction showed overall slower and less accurate responses as well as increased reaction times for negative versus neutral words compared with BPD patients without dissociation induction. Moreover, they showed comparatively decreased neuronal activity in the fusiform gyrus and parietal cortices independent of valence, but elevated activity in the left inferior frontal gyrus in response to negative versus neutral words. In conclusion, experimentally induced dissociation in BPD was associated with inefficient cognitive inhibition, particularly of negative stimuli, in the emotional Stroop task.

Trauma sequelae and cortisol levels in women exposed to intimate partner violence.

Intimate partner violence (IPV) is often a chronic form of trauma with deleterious mental health problems. Furthermore, IPV survivors have also often experienced trauma in childhood. Consequently, by examining a sample of IPV survivors, this study sought to assess typical trauma sequelae--Posttraumatic Stress Disorder (PTSD), Major Depressive Disorder (MDD), or dissociative symptoms-and trauma-related characteristics consistent with Herman's Trauma theory (1992; i.e., chronicity of trauma, age of first trauma exposure, and social support), in relation to hypothalamic-pituitary-adrenal axis function. The study compared basal and diurnal cortisol in women (n = 88) based on diagnostic status and symptom severity (PTSD [n = 14], PTSD and comorbid MDD [n = 43], subthreshold symptoms of PTSD and MDD [n = 19]), dissociative symptoms, and the aforementioned trauma-related characteristics to a matched control group (n = 12) without any lifetime history of mental health diagnoses or exposure to interpersonal trauma. Regardless of their diagnostic status and trauma-related characteristics, trauma-exposed women had higher levels of dissociative symptoms relative to women in the control group, and these dissociative symptoms were inversely related to awakening cortisol levels. Findings suggest that low cortisol levels may not be a diagnostic marker, but instead may be associated with a dissociative coping style developed in the context of trauma exposure, consistent with mechanisms posited by Trauma theory.

Phasic D1 and tonic D2 dopamine receptor signaling double dissociate the motivational effects of acute nicotine and chronic nicotine withdrawal.

Nicotine, the main psychoactive ingredient of tobacco smoke, induces negative motivational symptoms during withdrawal that contribute to relapse in dependent individuals. The neurobiological mechanisms underlying how the brain signals nicotine withdrawal remain poorly understood. Using electrophysiological, genetic, pharmacological, and behavioral methods, we demonstrate that tonic but not phasic activity is reduced during nicotine withdrawal in ventral tegmental area dopamine (DA) neurons, and that this pattern of signaling acts through DA D2 and adenosine A2A, but not DA D1, receptors. Selective blockade of phasic DA activity prevents the expression of conditioned place aversions to a single injection of nicotine in nondependent mice, but not to withdrawal from chronic nicotine in dependent mice, suggesting a shift from phasic to tonic dopaminergic mediation of the conditioned motivational response in nicotine dependent and withdrawn animals. Either increasing or decreasing activity at D2 or A2A receptors prevents the aversive motivational response to withdrawal from chronic nicotine, but not to acute nicotine. Modification of D1 receptor activity prevents the aversive response to acute nicotine, but not to nicotine withdrawal. This double dissociation demonstrates that the specific pattern of tonic DA activity at D2 receptors is a key mechanism in signaling the motivational effects experienced during nicotine withdrawal, and may represent a unique target for therapeutic treatments for nicotine addiction.

Posttraumatic oxytocin dysregulation: is it a link among posttraumatic self disorders, posttraumatic stress disorder, and pelvic visceral dysregulation conditions in women?

This article explicates a theory that oxytocin, a sexually dimorphic neurotransmitter and paracrine hormone, is a plausible mechanism linking early relational trauma with posttraumatic self disorders (e.g., dissociation, somatization, and interpersonal sensitivity), posttraumatic stress disorder, and pelvic visceral dysregulation disorders (e.g., irritable bowel syndrome, chronic pelvic pain, interstitial cystitis, and hyperemesis gravidarum). This posttraumatic oxytocin dysregulation disorders theory is consistent with the historical and contemporary literature. It integrates attention to psychological and physical comorbidities and could account for the increased incidence of these disorders among females. Specific propositions are explored in data from studies of traumatic stress and women's health.

[Evolution of dissociative learning].

This review considers data obtained during the entire research period of state-dependent learning. Understanding of this phenomenon has significantly evolved during the past decades, as a result of the increasing amount of facts revealed while studying state-dependent learning. Consequently, a situation has arisen where different papers may describe same phenomena using different terms. This does not promote understanding of the described phenomena. Therefore a need for a paper emerged, that would analyze the evolution of state-dependent learning and would offer terminology corresponding to all the data collected on the subject.

Dissociation of functional status from accrual of CML and RAGE in the aged mouse brain.

The objectives of this study were: (i) to identify regions of the aged mouse brain in which advanced glycation end-products (AGEs) were increased, and (ii) assess the functional significance of AGEs by determining the extent to which they could predict age-related brain dysfunction. Densitometric analyses of immunoblots for N epsilon-(carboxymethyl)lysine (CML), a predominant AGE, and receptor for AGE (RAGE), were performed in different brain regions of mice aged 8 or 25 months. The 25-month-old mice were tested for ability to perform on tests of cognitive and psychomotor function prior to assessment of CML or RAGE, to determine if immunostaining results could predict functional impairment among the older mice. The amounts of CML increased with age in cortex, hippocampus, striatum, and midbrain, but were unchanged in the brainstem and cerebellum. Increases in RAGE were evident in all brain regions but the hippocampus, and were not linked to increased amounts of CML. Different statistical approaches each failed to reveal any strong association between the degree of age-related functional impairment among individual mice and amounts of CML or RAGE in any particular region of the brain. The findings from this study suggest that accrual of CML and expression of RAGE in different brain regions are time-related phenomena that do not account for individual differences in brain aging or cognitive decline.

Investigating the possible effects of trauma experiences and 5-HTT on the dissociative experiences of patients with OCD using path analysis and multiple regression.

Dissociation is defined as the disruption of the usually integrated functions of consciousness, such as memory, identity, and perceptions of the environment. Causes include various psychological, neurological and neurobiological mechanisms, none of which have been consistently supported. To our knowledge, the role of gene-environment interactions in dissociative experiences in obsessive-compulsive disorder (OCD) has not previously been investigated. Eighty-three Caucasian patients (29 male, 54 female) with a principal diagnosis of OCD were included. The Dissociative Experiences Scale was used to assess dissociation. The role of childhood trauma (assessed with the Childhood Trauma Questionnaire), and a functional 44-bp insertion/deletion polymorphism in the promoter region of the serotonin transporter, or 5-HTT, in mediating dissociation, was investigated using multiple regression analysis and path analysis using the partial least squares model. Both analyses indicated that an interaction between physical neglect and the S/S genotype of the 5-HTT gene significantly predicted dissociation in patients with OCD. Dissociation may be a predictor of poorer treatment outcome in patients with OCD; therefore, a better understanding of the mechanisms that underlie this phenomenon may be useful. Here, two different but related statistical techniques (multiple regression and partial least squares), confirmed that physical neglect and the 5-HTT genotype jointly play a role in predicting dissociation in OCD.

gamma-Aminobutyric acid-serotonin interactions in healthy men: implications for network models of psychosis and dissociation.

BACKGROUND: This study tested the hypothesis that deficits in gamma-aminobutyric acid type A (GABA(A)) receptor function might create a vulnerability to the psychotogenic and perceptual altering effects of serotonergic (5-HT(2A/2C)) receptor stimulation. The interactive effects of iomazenil, an antagonist and partial inverse agonist of the benzodiazepine site of the GABA(A) receptor complex, and m-chlorophenylpiperazine (m-CPP), a partial agonist of 5-HT(2A/2C) receptors, were studied in 23 healthy male subjects. METHODS: Subjects underwent 4 days of testing, during which they received intravenous infusions of iomazenil/placebo followed by m-CPP/placebo in a double-blind, randomized crossover design. Behavioral, cognitive, and hormonal data were collected before drug infusions and periodically for 200 min after. RESULTS: Iomazenil and m-CPP interacted in a synergistic manner to produce mild psychotic symptoms and perceptual disturbances without impairing cognition. Iomazenil and m-CPP increased anxiety in an additive fashion. Iomazenil and m-CPP interacted in a synergistic manner to increase serum cortisol. CONCLUSIONS: Gamma-aminobutyric acid-ergic deficits might increase the vulnerability to the psychotomimetic and perceptual altering effects of serotonergic agents. These data suggest that interactions between GABA(A) and 5-HT systems might contribute to the pathophysiology of psychosis and dissociative-like perceptual states.

Reduced glucose metabolism in temporo-parietal cortices of women with borderline personality disorder.

Individuals with borderline personality disorder (BPD) and posttraumatic stress disorder (PTSD) often experience dissociative symptoms. Evidence is increasing that stress-related hyperglutamatergic states may contribute to dissociative symptoms and neurodegeneration in temporo-parietal cortical areas. Seventeen young women with BPD who had been exposed to severe childhood physical/sexual abuse and presented with pronounced dissociative symptoms underwent (18)fluoro-2-deoxyglucose positron emission tomography (FDG-PET). Nine healthy, matched volunteers served as comparison subjects. Borderline subjects displayed reduced FDG uptake (as analyzed by SPM) in the right temporal pole/anterior fusiform gyrus and in the left precuneus and posterior cingulate cortex. Impaired memory performance among borderline subjects was significantly correlated with metabolic activity in ventromedial and lateral temporal cortices. Our results demonstrate regional hypometabolism in temporal and medial parietal cortical regions known to be involved in episodic memory consolidation and retrieval. Currently, the precuneus/posterior cingulate cortex is modeled as part of a network of tonically active brain regions that continuously gather information about the world around and within us. Decreased resting metabolic rate of these regions may reflect dissociative symptoms and possibly also identity disturbances and interpersonal difficulties of individuals with BPD.

Dissociation in conditioned dopamine release in the nucleus accumbens core and shell in response to cocaine cues and during cocaine-seeking behavior in rats.

The dopaminergic innervation of the nucleus accumbens is generally agreed to mediate the primary reinforcing and locomotor effects of psychostimulants, but there is less consensus on conditioned dopamine (DA) release during drug-seeking behavior. We investigated the neurochemical correlates of drug-seeking behavior under the control of a drug-associated cue [a light conditioned stimulus (CS+)] and to noncontingent presentations of the CS+ in the core and shell subregions of the nucleus accumbens. Rats self-administered cocaine under a continuous reinforcement schedule in which a response on one of two identical levers led to an intravenous cocaine infusion (0.25 mg/infusion) and a 20 sec light CS+. Response requirements for cocaine and the CS+ were then progressively increased until stable responding was established under a second-order schedule of reinforcement. During microdialysis, rats were presented noncontingently with a set of 10 sec CS+ and neutral tone stimuli (CS-) before and after a 90 min period during which they responded for cocaine under a second-order schedule. Results showed the following: (1) nucleus accumbens DA increased in both the core and shell during intravenous cocaine self-administration; (2) noncontingent presentations of a cocaine-associated CS+ led to increased DA release selectively in the nucleus accumbens core; and (3) extracellular DA levels were unaltered in both core and shell during a protracted period of drug-seeking behavior under the control of the same cocaine-associated cue. These results indicate that the mesolimbic dopamine system is activated after exposure to drug-associated stimuli under specific conditions.

[The phosphorylation of synaptic membrane proteins in the occurrence of prolonged dissociative states induced by carbacholine]. / Fosforilirovanie belkov sinapticheskikh membran pri vozniknovenii dlitel'nykh dissotsiirovannykh sostoianii, vyzvannykh karbakholinom.

Prolonged dissociated state of rats resulting from carbaholine stimulation of m-cholinoreceptors was studied by determining phosphorylation of synaptic membranes proteins. Unusually strong and prolonged activation was found in the process of phosphorylation of the hippocampus synaptic proteins under the influence of single carbaholine administration. Conclusion is made about direct participation of the hippocampus in mechanisms of memory dissociation and also about perspectiveness of the applied method for functional characteristics of separate brain structures.