P11 deficiency increases stress reactivity along with HPA axis and autonomic hyperresponsiveness.

Patients suffering from mood disorders and anxiety commonly exhibit hypothalamic-pituitary-adrenocortical (HPA) axis and autonomic hyperresponsiveness. A wealth of data using preclinical animal models and human patient samples indicate that p11 deficiency is implicated in depression-like phenotypes. In the present study, we used p11-deficient (p11KO) mice to study potential roles of p11 in stress responsiveness. We measured stress response using behavioral, endocrine, and physiological readouts across early postnatal and adult life. Our data show that p11KO pups respond more strongly to maternal separation than wild-type pups, even though their mothers show no deficits in maternal behavior. Adult p11KO mice display hyperactivity of the HPA axis, which is paralleled by depression- and anxiety-like behaviors. p11 was found to be highly enriched in vasopressinergic cells of the paraventricular nucleus and regulates HPA hyperactivity in a V1B receptor-dependent manner. Moreover, p11KO mice display sympathetic-adrenal-medullary (SAM) axis hyperactivity, with elevated adrenal norepinephrine and epinephrine levels. Using conditional p11KO mice, we demonstrate that this SAM hyperactivity is partially regulated by the loss of p11 in serotonergic neurons of the raphe nuclei. Telemetric electrocardiogram measurements show delayed heart rate recovery in p11KO mice in response to novelty exposure and during expression of fear following auditory trace fear conditioning. Furthermore, p11KO mice have elevated basal heart rate in fear conditioning tests indicating increased autonomic responsiveness. This set of experiments provide strong and versatile evidence that p11 deficiency leads to HPA and SAM axes hyperresponsiveness along with increased stress reactivity.

Presynaptic inhibition upon CB1 or mGlu2/3 receptor activation requires ERK/MAPK phosphorylation of Munc18-1.

Presynaptic cannabinoid (CB1R) and metabotropic glutamate receptors (mGluR2/3) regulate synaptic strength by inhibiting secretion. Here, we reveal a presynaptic inhibitory pathway activated by extracellular signal-regulated kinase (ERK) that mediates CB1R- and mGluR2/3-induced secretion inhibition. This pathway is triggered by a variety of events, from foot shock-induced stress to intense neuronal activity, and induces phosphorylation of the presynaptic protein Munc18-1. Mimicking constitutive phosphorylation of Munc18-1 results in a drastic decrease in synaptic transmission. ERK-mediated phosphorylation of Munc18-1 ultimately leads to degradation by the ubiquitin-proteasome system. Conversely, preventing ERK-dependent Munc18-1 phosphorylation increases synaptic strength. CB1R- and mGluR2/3-induced synaptic inhibition and depolarization-induced suppression of excitation (DSE) are reduced upon ERK/MEK pathway inhibition and further reduced when ERK-dependent Munc18-1 phosphorylation is blocked. Thus, ERK-dependent Munc18-1 phosphorylation provides a major negative feedback loop to control synaptic strength upon activation of presynaptic receptors and during intense neuronal activity.

Seizures and disturbed brain potassium dynamics in the leukodystrophy megalencephalic leukoencephalopathy with subcortical cysts.

OBJECTIVE: Loss of function of the astrocyte-specific protein MLC1 leads to the childhood-onset leukodystrophy "megalencephalic leukoencephalopathy with subcortical cysts" (MLC). Studies on isolated cells show a role for MLC1 in astrocyte volume regulation and suggest that disturbed brain ion and water homeostasis is central to the disease. Excitability of neuronal networks is particularly sensitive to ion and water homeostasis. In line with this, reports of seizures and epilepsy in MLC patients exist. However, systematic assessment and mechanistic understanding of seizures in MLC are lacking. METHODS: We analyzed an MLC patient inventory to study occurrence of seizures in MLC. We used two distinct genetic mouse models of MLC to further study epileptiform activity and seizure threshold through wireless extracellular field potential recordings. Whole-cell patch-clamp recordings and K+ -sensitive electrode recordings in mouse brain slices were used to explore the underlying mechanisms of epilepsy in MLC. RESULTS: An early onset of seizures is common in MLC. Similarly, in MLC mice, we uncovered spontaneous epileptiform brain activity and a lowered threshold for induced seizures. At the cellular level, we found that although passive and active properties of individual pyramidal neurons are unchanged, extracellular K+ dynamics and neuronal network activity are abnormal in MLC mice. INTERPRETATION: Disturbed astrocyte regulation of ion and water homeostasis in MLC causes hyperexcitability of neuronal networks and seizures. These findings suggest a role for defective astrocyte volume regulation in epilepsy. Ann Neurol 2018;83:636-649.

Protein instability, haploinsufficiency, and cortical hyper-excitability underlie STXBP1 encephalopathy.

De novo heterozygous mutations in STXBP1/Munc18-1 cause early infantile epileptic encephalopathies (EIEE4, OMIM #612164) characterized by infantile epilepsy, developmental delay, intellectual disability, and can include autistic features. We characterized the cellular deficits for an allelic series of seven STXBP1 mutations and developed four mouse models that recapitulate the abnormal EEG activity and cognitive aspects of human STXBP1-encephalopathy. Disease-causing STXBP1 variants supported synaptic transmission to a variable extent on a null background, but had no effect when overexpressed on a heterozygous background. All disease variants had severely decreased protein levels. Together, these cellular studies suggest that impaired protein stability and STXBP1 haploinsufficiency explain STXBP1-encephalopathy and that, therefore, Stxbp1+/- mice provide a valid mouse model. Simultaneous video and EEG recordings revealed that Stxbp1+/- mice with different genomic backgrounds recapitulate the seizure/spasm phenotype observed in humans, characterized by myoclonic jerks and spike-wave discharges that were suppressed by the antiepileptic drug levetiracetam. Mice heterozygous for Stxbp1 in GABAergic neurons only, showed impaired viability, 50% died within 2-3 weeks, and the rest showed stronger epileptic activity. c-Fos staining implicated neocortical areas, but not other brain regions, as the seizure foci. Stxbp1+/- mice showed impaired cognitive performance, hyperactivity and anxiety-like behaviour, without altered social behaviour. Taken together, these data demonstrate the construct, face and predictive validity of Stxbp1+/- mice and point to protein instability, haploinsufficiency and imbalanced excitation in neocortex, as the underlying mechanism of STXBP1-encephalopathy. The mouse models reported here are valid models for development of therapeutic interventions targeting STXBP1-encephalopathy.

A multiscale entropy-based tool for scoring severity of systemic inflammation.

OBJECTIVE: Early detection and start of appropriate treatment are highly correlated with survival of sepsis and septic shock, but the currently available predictive tools are not sensitive enough to identify patients at risk. DESIGN: Linear (time and frequency domain) and nonlinear (unifractal and multiscale complexity dynamics) measures of beat-to-beat interval variability were analyzed in two mouse models of inflammatory shock to determine if they are sensitive enough to predict outcome. SETTING: University research laboratory. SUBJECTS: Blood pressure transmitter-implanted female C57BL/6J mice. INTERVENTIONS: IV administration of tumor necrosis factor (n = 11) or lipopolysaccharide (n = 14). MEASUREMENTS AND MAIN RESULTS: Contrary to linear indices of variability, unifractal dynamics, and absolute heart rate or blood pressure, quantification of complex beat-to-beat dynamics using multiscale entropy was able to predict survival outcome starting as early as 40 minutes after induction of inflammatory shock. Based on these results, a new and clinically relevant index of multiscale entropy was developed that scores the key features of a multiscale entropy profile. Contrary to multiscale entropy, multiscale entropy scoring can be followed as a function of time to monitor disease progression with limited loss of information. CONCLUSIONS: Analysis of multiscale complexity of beat-to-beat dynamics at high temporal resolution has potential as a sensitive prognostic tool with translational power that can predict survival outcome in systemic inflammatory conditions such as sepsis and septic shock.

Blunted autonomic reactivity to pharmacological panic challenge under long-term escitalopram treatment in healthy men.

BACKGROUND: Central serotonergic pathways influence brain areas involved in vagal cardiovascular regulation and, thereby, influence sympathetic efferent activity. Selective serotonin reuptake inhibitors (SSRIs) affect multiple serotonergic pathways, including central autonomic pathways. However, only a few studies have assessed SSRI-mediated effects on autonomic reactivity in healthy individuals using heart rate variability (HRV). METHODS: The present study assessed the influence of long-term treatment with escitalopram (ESC) on autonomic reactivity to an intravenous application of 50 µg cholecystokinin tetrapeptide (CCK-4) in 30 healthy young men using a double-blind, placebo (PLA)-controlled, randomized, within-subject cross-over design. Main outcome measures were time- and frequency-domain HRV parameters, assessed at both baseline and immediately after CCK-4 application. RESULTS: Results showed substantial effects for the treatment × CCK-4 challenge interaction with respect to heart rate (p < 0.001; pη(2) = 0.499), SDNN (p < 0.001; pη(2) = 576), RMSSD (p = 0.015; pη(2) = 194), NN50% (p = 0.008; pη(2) = 0.224), and LF% (p = 0.014; pη(2) = 0.196), and moderate effects with respect HF% (p = 0.099; pη(2) = 0.094), with PLA subjects showing a higher increase in HR and SDNN and a higher decrease in RMSSD, NN50, LF and HF than subjects in the ESC condition. Thus, ESC treatment significantly blunted the autonomic reactivity to CCK-4. Secondary analysis indicated no effect of the 5-HTTLPR polymorphism on CCK-4-induced autonomic response. CONCLUSIONS: Our results support findings suggesting an effect of SSRI treatment on autonomic regulation and provide evidence that ESC treatment is associated with blunted autonomic reactivity in healthy men.

Continuous locomotor activity monitoring to assess animal welfare following intracranial surgery in mice.

Locomotor activity can serve as a readout to identify discomfort and pain. Therefore, monitoring locomotor activity following interventions that induce potential discomfort may serve as a reliable method for evaluating animal health, complementing conventional methods such as body weight measurement. In this study, we used the digital ventilated cage (DVC®) system for the assessment of circadian locomotor activity, in addition to body weight monitoring, following intracranial stereotaxic surgery in an Alzheimer's disease mouse model (C57BL/6J/APPswe/PSEN1dE9). Stereotaxic surgery did not affect the organization of circadian locomotor activity of both 7-8-week-old and 19-21-week-old mice. However, we observed that both young and old mice exhibited a significant decrease in activity during the dark phase. Also, our study shows that changes in locomotor activity exhibit higher sensitivity in detecting alterations indicative of animal health compared to measuring body weight. In contrast to 7-8-week-old mice, where we observed no genotypic differences in locomotor activity, 19-21-week-old APP/PS1 mice showed increased locomotor activity compared to wild-type mice. Furthermore, our analyses revealed that a subset of the 7-8-week-old mice showed increased locomotor activity during the initial peak of the dark phase. One mouse experienced sudden death early in life, possibly due to epileptic seizures. Altogether, our findings affirm continuous activity measurements as used in the DVC® as a highly valuable objective method for post-surgical welfare monitoring. Its discerning capacity not only facilitates circadian locomotor rhythm assessment but also enables the identification of individual aberrant activity patterns, possibly indicative of epileptic seizures.

Diminished vagal activity and blunted diurnal variation of heart rate dynamics in posttraumatic stress disorder.

Affected autonomic heart regulation is implicated in the pathophysiology of cardiovascular diseases and is associated with posttraumatic stress disorder (PTSD). However, although sympathetic hyperactivation has been repeatedly shown in PTSD, research has neglected parasympathetic function. The objective of this study is the long-term assessment of heart rate (HR) dynamics and its diurnal changes as an index of autonomic imbalance in PTSD. Since tonic parasympathetic activity underlies long-range correlation of heartbeat interval fluctuations in the healthy state, we included nonlinear (unifractal) analysis as an important and sensitive readout to assess functional alterations. We conducted electrocardiogram recordings over a 24-h period in 15 deployed male subjects with moderate to high levels of combat exposure (PTSD: n = 7; combat controls: n = 8) in the supine position. HR dynamics were assessed in two 5-h sub-epochs in the time and frequency domains, and by nonlinear analysis based on detrended fluctuation analysis. Psychiatric symptoms were assessed using structured interviews, including the Clinician Administered PTSD Scale. Subjects with PTSD showed significantly higher baseline HR, higher LF/HF ratio in the frequency domain, blunted differences between day and night-time measures, as well as a higher scaling coefficient αfast during the day, indicating diminished tonic parasympathetic activity. Diminished diurnal differences and blunted tonic parasympathetic activity altering HR dynamics suggest central neuroautonomic dysregulation that could represent a possible link to increased cardiovascular disease in PTSD.

Heart Rate Variability as a Translational Dynamic Biomarker of Altered Autonomic Function in Health and Psychiatric Disease.

The autonomic nervous system (ANS) is responsible for the precise regulation of tissue functions and organs and, thus, is crucial for optimal stress reactivity, adaptive responses and health in basic and challenged states (survival). The fine-tuning of central ANS activity relies on the internal central autonomic regulation system of the central autonomic network (CAN), while the peripheral activity relies mainly on the two main and interdependent peripheral ANS tracts, the sympathetic nervous system (SNS) and the parasympathetic nervous system (PNS). In disease, autonomic imbalance is associated with decreased dynamic adaptability and increased morbidity and mortality. Acute or prolonged autonomic dysregulation, as observed in stress-related disorders, affects CAN core centers, thereby altering downstream peripheral ANS function. One of the best established and most widely used non-invasive methods for the quantitative assessment of ANS activity is the computerized analysis of heart rate variability (HRV). HRV, which is determined by different methods from those used to determine the fluctuation of instantaneous heart rate (HR), has been used in many studies as a powerful index of autonomic (re)activity and an indicator of cardiac risk and ageing. Psychiatric patients regularly show altered autonomic function with increased HR, reduced HRV and blunted diurnal/circadian changes compared to the healthy state. The aim of this article is to provide basic knowledge on ANS function and (re)activity assessment and, thus, to support a much broader use of HRV as a valid, transdiagnostic and fully translational dynamic biomarker of stress system sensitivity and vulnerability to stress-related disorders in neuroscience research and clinical psychiatric practice. In particular, we review the functional levels of central and peripheral ANS control, the main neurobiophysiologic theoretical models (e.g., polyvagal theory, neurovisceral integration model), the precise autonomic influence on cardiac function and the definition and main aspects of HRV and its different measures (i.e., time, frequency and nonlinear domains). We also provide recommendations for the proper use of electrocardiogram recordings for HRV assessment in clinical and research settings and highlight pathophysiological, clinical and research implications for a better functional understanding of the neural and molecular mechanisms underlying healthy and malfunctioning brain-heart interactions in individual stress reactivity and psychiatric disorders.

A mouse model of high trait anxiety shows reduced heart rate variability that can be reversed by anxiolytic drug treatment.

Increasing evidence suggests that specific physiological measures may serve as biomarkers for successful treatment to alleviate symptoms of pathological anxiety. Studies of autonomic function investigating parameters such as heart rate (HR), HR variability and blood pressure (BP) indicated that HR variability is consistently reduced in anxious patients, whereas HR and BP data show inconsistent results. Therefore, HR and HR variability were measured under various emotionally challenging conditions in a mouse model of high innate anxiety (high anxiety behaviour; HAB) vs. control normal anxiety-like behaviour (NAB) mice. Baseline HR, HR variability and activity did not differ between mouse lines. However, after cued Pavlovian fear conditioning, both elevated tachycardia and increased fear responses were observed in HAB mice compared to NAB mice upon re-exposure to the conditioning stimulus serving as the emotional stressor. When retention of conditioned fear was tested in the home cage, HAB mice again displayed higher fear responses than NAB mice, while the HR responses were similar. Conversely, in both experimental settings HAB mice consistently exhibited reduced HR variability. Repeated administration of the anxiolytic NK1 receptor antagonist L-822429 lowered the conditioned fear response and shifted HR dynamics in HAB mice to a more regular pattern, similar to that in NAB mice. Additional receiver-operating characteristic (ROC) analysis demonstrated the high specificity and sensitivity of HR variability to distinguish between normal and high anxiety trait. These findings indicate that assessment of autonomic response in addition to freezing might be a useful indicator of the efficacy of novel anxiolytic treatments.