Type 1 melanocortin receptors in pro-opiomelanocortin-, vasopressin-, and oxytocin-immunopositive neurons in different areas of mouse brain.

Information on the localization of the Type 1 melanocortin receptors (MC1Rs) in different regions of the brain is very scarce. As a result, the role of MC1Rs in the functioning of brain neurons and in the central regulation of physiological functions has not been studied. This work aimed to study the expression and distribution of MС1Rs in different brain areas of female C57Bl/6J mice. Using real-time polymerase chain reaction, we demonstrated the Mс1R gene expression in the cerebral cortex, midbrain, hypothalamus, medulla oblongata, and hippocampus. Using an immunohistochemical approach, we showed the MС1R localization in neurons of the hypothalamic arcuate, paraventricular and supraoptic nuclei, nucleus tractus solitarius (NTS), dorsal hippocampus, substantia nigra, and cerebral cortex. Using double immunolabeling, the MC1Rs were visualized on the surface and in the bodies and outgrowths of pro-opiomelanocortin (POMC)-immunopositive neurons in the hypothalamic arcuate nucleus, NTS, hippocampal CA3 and CA1 regions, and cerebral cortex. Co-localization with POMC indicates that MC1R, like MC3R, is able to function as an autoreceptor. In the paraventricular and supraoptic nuclei, MC1Rs were visualized on the surface and in the cell bodies of vasopressin- and oxytocin-immunopositive neurons, indicating a relationship between hypothalamic MC1R signaling and vasopressin and oxytocin production. The data obtained indicate a wide distribution of MC1Rs in different areas of the mouse brain and their localization in POMC-, vasopressin- and oxytocin-immunopositive neurons, which may indicate the participation of MC1Rs in the control of many physiological processes in the central nervous system.

The effects of chronic restraint stress on empathy-like behaviour in rats.

It is clearly known that psychological stress is an important threat to health in today's modern societies. Recent studies have shown that acute stress causes an increase in positive social behaviours such as prosocial behaviour and devotion which are components of empathic behaviour. Neuropsychiatric manifestations such as anxiety and depression may affect empathic behaviour. The aim of this study was to investigate the effects of chronic restraint stress on empathy-like behaviour and the histopathological changes in the amygdala, prefrontal cortex in the adrenal glands and thymus, as well as the neurochemical pathways associated with empathy, oxytocin and vasopressin. The chronic stress group was subjected to restraint stress daily for 14 days after all subjects were trained to rescue its stressed cagemate using empathy test equipment for 12 days. It was observed that chronic restraint stress had no effect on empathy-like behaviour in rats. Vasopressin levels in amygdala was increased in chronic stress group compared to control group. Anxiety and depression indicators did not change in both groups. In the open field test, control group spent more time in thigmo zone compared to chronic stress group. Adrenal glands relative weights and apoptotic cell ratios were significantly higher in the chronic stress group compared to the control group (expectedly). Although there was no significant difference in behavioral tests, histopathological changes were detected. In subsequent studies, it is appropriate to examine the effects of different types of stress applications, gender-related changes, and other neurochemical pathways associated with stress and empathy.

The effect of amisulpride, olanzapine, quetiapine, and aripiprazole single administration on c-Fos expression in vasopressinergic and oxytocinergic neurons of the rat hypothalamic paraventricular nucleus.

Antipsychotics, including amisulpride (AMI), quetiapine (QUE), aripiprazole (ARI), and olanzapine (OLA), are used to treat mental illnesses associated with psychotic symptoms. The effect of these drugs on c-Fos expression in vasopressinergic (AVP) and oxytocinergic (OXY) neurons was studied in the hypothalamic paraventricular nucleus (PVN) of rats. The presence of c-Fos in AVP and OXY perikarya was investigated in seven PVN cells segregations: the anterior (Ant), dorsal cup (Dc), wing-shaped (Wi), periventricular zone (Pe), circle-shaped core (Co) and shell of core (Sh), and the posterior (pPVN) after an acute treatment with AMI-20 mg/kg, QUE-15 mg/kg, ARI-10 mg/kg, and OLA-5 mg/kg/bw in rats. Ninety min after treatments, the animals were sacrificed by transcardial perfusion with fixative and the PVN area sliced into 35 µm thick coronal sections for immunohistochemistry. The c-Fos was processed by avidin-biotin-peroxidase complex intensified with nickel-enhanced 3,3'-diaminobenzidine tetrahydrochloride. Visualization of AVP- and OXY-synthesizing neurons was achieved by a fluorescent marker Alexa Flour 568. The c-Fos-AVP and c-Fos-OXY colocalizations were evaluated from c-Fos stained sections merged with AVP or OXY ones. AMI, QUE, ARI, and OLA, single administration distinctly increased the c-Fos expression in each of the PVN cells segregations. QUE induced the highest magnitude of activation of AVP and OXY neurons, while OLA and AMI had only moderate effects. Incontestable variabilities detected in c-Fos expression in PVN AVP and OXY neurons extend the knowledge of selected antipsychotics extra-striatal actions and may also be helpful in a presumption of their possible functional impact.

Ultrasensitive and Highly Selective Graphene-Based Field-Effect Transistor Biosensor for Anti-Diuretic Hormone Detection.

Nephrogenic diabetes insipidus (NDI), which can be congenital or acquired, results from the failure of the kidney to respond to the anti-diuretic hormone (ADH). This will lead to excessive water loss from the body in the form of urine. The kidney, therefore, has a crucial role in maintaining water balance and it is vital to restore this function in an artificial kidney. Herein, an ultrasensitive and highly selective aptameric graphene-based field-effect transistor (GFET) sensor for ADH detection was developed by directly immobilizing ADH-specific aptamer on a surface-modified suspended graphene channel. This direct immobilization of aptamer on the graphene surface is an attempt to mimic the functionality of collecting tube V 2 receptors in the ADH biosensor. This aptamer was then used as a probe to capture ADH peptide at the sensing area which leads to changes in the concentration of charge carriers in the graphene channel. The biosensor shows a significant increment in the relative change of current ratio from 5.76 to 22.60 with the increase of ADH concentration ranging from 10 ag/mL to 1 pg/mL. The ADH biosensor thus exhibits a sensitivity of 50.00 µA· ( g / mL ) - 1 with a limit of detection as low as 3.55 ag/mL. In specificity analysis, the ADH biosensor demonstrated a higher current value which is 338.64 µA for ADH-spiked in phosphate-buffered saline (PBS) and 557.89 µA for ADH-spiked in human serum in comparison with other biomolecules tested. This experimental evidence shows that the ADH biosensor is ultrasensitive and highly selective towards ADH in PBS buffer and ADH-spiked in human serum.

Neonatal CSF vasopressin concentration predicts later medical record diagnoses of autism spectrum disorder.

Autism spectrum disorder (ASD) is a brain disorder characterized by social impairments. ASD is currently diagnosed on the basis of behavioral criteria because no robust biomarkers have been identified. However, we recently found that cerebrospinal fluid (CSF) concentration of the "social" neuropeptide arginine vasopressin (AVP) is significantly lower in pediatric ASD cases vs. controls. As an initial step in establishing the direction of causation for this association, we capitalized upon a rare biomaterials collection of newborn CSF samples to conduct a quasi-prospective test of whether this association held before the developmental period when ASD first manifests. CSF samples had been collected in the course of medical care of 0- to 3-mo-old febrile infants (n = 913) and subsequently archived at -70 °C. We identified a subset of CSF samples from individuals later diagnosed with ASD, matched them 1:2 with appropriate controls (n = 33 total), and quantified their AVP and oxytocin (OXT) concentrations. Neonatal CSF AVP concentrations were significantly lower among ASD cases than controls and individually predicted case status, with highest precision when cases with comorbid attention-deficit/hyperactivity disorder were removed from the analysis. The associations were specific to AVP, as ASD cases and controls did not differ in neonatal CSF concentrations of the structurally related neuropeptide, OXT. These preliminary findings suggest that a neurochemical marker of ASD may be present very early in life, and if replicated in a larger, prospective study, this approach could transform how ASD is detected, both in behaviorally symptomatic children, and in infants at risk for developing it.

Differences in the number of oxytocin, vasopressin, and tyrosine hydroxylase cells in brain regions associated with mating among great, midday, and Mongolian gerbils.

Neurotransmitters, such as oxytocin (OT), vasopressin (AVP), and dopamine (DA), within the mesolimbic system have deeply conserved roles in regulating mating-related behaviors. However, comparative studies among monogamous and polygamous animals focus mainly on Microtus; very little research has been done in gerbils. Here, we measured body weight, body length, tail length, serum hormone concentrations, and the immunoreactive (ir)-cells of OT, AVP, and tyrosine hydroxylase (TH) in the brain of the polygamous great gerbil (Rhombomys opimus), midday gerbil (Meriones meridianus), and monogamous Mongolian gerbil (Meriones unguiculatus). Body weight, body length, tail length, and serum AVP concentrations were greater in the great gerbil than in the midday gerbil and Mongolian gerbil. The number of OT and AVP cells in the para ventricular nucleus (PVN) and supra optic nucleus (SON) of the hypothalamus were greater in the Mongolian gerbil than in the great gerbil and midday gerbil. Similarly, the number of TH cells in the PVN, medial preoptic area (MPOA), and ventral tegmental area (VTA) was greater in the Mongolian gerbil than in the great gerbil and midday gerbil. To summarize, the number of OT and AVP cells in the PVN and SON and TH cells in the PVN, MPOA, and VTA in the monogamous Mongolian gerbil are greater than those in the great gerbil and midday gerbil.

Exogenous vasopressin dose-dependently modulates gastric microcirculatory oxygenation in dogs via V1A receptor.

BACKGROUND: Hypercapnia improves gastric microcirculatory oxygenation (µHbO2) and increases vasopressin plasma levels, whereas V1A receptor blockade abolishes the increase of µHbO2. The aim of this study was to evaluate the effect of exogenous vasopressin (AVP) in increasing doses on microcirculatory perfusion and oxygenation and systemic hemodynamic variables. Furthermore, we evaluated the role of the vasopressin V1A receptor in mediating the effects. METHODS: In repetitive experiments, six anesthetized dogs received a selective vasopressin V1A receptor inhibitor ([Pmp1, Tyr (Me)2]-Arg8-Vasopressin) or sodium chloride (control groups). Thereafter, a continuous infusion of AVP was started with dose escalation every 30 min (0.001 ng/kg/min-1 ng/kg/min). Microcirculatory variables of the oral and gastric mucosa were measured with reflectance spectrometry, laser Doppler flowmetry, and incident dark field imaging. Transpulmonary thermodilution was used to measure systemic hemodynamic variables. AVP plasma concentrations were measured during baseline conditions and 30 min after each dose escalation. RESULTS: During control conditions, gastric µHbO2 did not change during the course of experiments. Infusion of 0.001 ng/kg/min and 0.01 ng/kg/min AVP increased gastric µHbO2 to 87 ± 4% and 87 ± 6%, respectively, compared to baseline values (80 ± 7%), whereas application of 1 ng/kg/min AVP strongly reduced gastric µHbO2 (59 ± 16%). V1A receptor blockade prior to AVP treatment abolished these effects on µHbO2. AVP dose-dependently enhanced systemic vascular resistance (SVR) and decreased cardiac output (CO). After prior V1A receptor blockade, SVR was reduced and CO increased (0.1 ng/kg/min + 1 ng/kg/min AVP). CONCLUSIONS: Exogenous AVP dose-dependently modulates gastric µHbO2, with an increased µHbO2 with ultra-low dose AVP. The effects of AVP on µHbO2 are abolished by V1A receptor inhibition. These effects are independent of a modulation of systemic hemodynamic variables.

Diabetes insipidus.

Diabetes insipidus (DI) is a disorder characterized by excretion of large amounts of hypotonic urine. Central DI results from a deficiency of the hormone arginine vasopressin (AVP) in the pituitary gland or the hypothalamus, whereas nephrogenic DI results from resistance to AVP in the kidneys. Central and nephrogenic DI are usually acquired, but genetic causes must be evaluated, especially if symptoms occur in early childhood. Central or nephrogenic DI must be differentiated from primary polydipsia, which involves excessive intake of large amounts of water despite normal AVP secretion and action. Primary polydipsia is most common in psychiatric patients and health enthusiasts but the polydipsia in a small subgroup of patients seems to be due to an abnormally low thirst threshold, a condition termed dipsogenic DI. Distinguishing between the different types of DI can be challenging and is done either by a water deprivation test or by hypertonic saline stimulation together with copeptin (or AVP) measurement. Furthermore, a detailed medical history, physical examination and imaging studies are needed to ensure an accurate DI diagnosis. Treatment of DI or primary polydipsia depends on the underlying aetiology and differs in central DI, nephrogenic DI and primary polydipsia.

EJE AWARD 2019: New diagnostic approaches for patients with polyuria polydipsia syndrome.

Diabetes insipidus (DI), be it from central or nephrogenic origin, must be differentiated from secondary forms of hypotonic polyuria such as primary polydipsia. Differentiation is crucial since wrong treatment can have deleterious consequences. Since decades, the gold standard for differentiation has been the water deprivation test, which has limitations leading to an overall unsatisfying diagnostic accuracy. Furthermore, it is cumbersome for patients with a long test duration. Clinical signs and symptoms and MRI characteristics overlap between patients with DI and primary polydipsia. The direct test including vasopressin (AVP) measurement upon osmotic stimulation was meant to overcome these limitations, but failed to enter clinical practice mainly due to technical constraints of the AVP assay. Copeptin is secreted in equimolar amount to AVP but can easily be measured with a sandwich immunoassay. A high correlation between copeptin and AVP has been shown. Accordingly, copeptin mirrors the amount of AVP in the circulation and has led to a 'revival' of the direct test in the differential diagnosis of DI. We have shown that a baseline copeptin, without prior thirsting, unequivocally identifies patients with nephrogenic DI. In contrast, for the differentiation between central DI and primary polydipsia, a stimulated copeptin level of 4.9 pmol/L upon hypertonic saline infusion differentiates these two entities with a high diagnostic accuracy and is superior to the water deprivation test. Close sodium monitoring during the test is a prerequisite. Further new test methods are currently evaluated and might provide an even simpler way of differential diagnosis in the future.

Oxytocin/vasopressin-like peptide inotocin regulates cuticular hydrocarbon synthesis and water balancing in ants.

Oxytocin/vasopressin-like peptides are important regulators of physiology and social behavior in vertebrates. However, the function of inotocin, the homologous peptide in arthropods, remains largely unknown. Here, we show that the level of expression of inotocin and inotocin receptor are correlated with task allocation in the ant Camponotus fellah Both genes are up-regulated when workers age and switch tasks from nursing to foraging. in situ hybridization revealed that inotocin receptor is specifically expressed in oenocytes, which are specialized cells synthesizing cuticular hydrocarbons which function as desiccation barriers in insects and for social recognition in ants. dsRNA injection targeting inotocin receptor, together with pharmacological treatments using three identified antagonists blocking inotocin signaling, revealed that inotocin signaling regulates the expression of cytochrome P450 4G1 (CYP4G1) and the synthesis of cuticular hydrocarbons, which play an important role in desiccation resistance once workers initiate foraging.

Oestradiol acts through its beta receptor to increase vasopressin neuronal activation and secretion induced by dehydration.

Vasopressinergic neurones of the supraoptic (SON) and paraventricular (PVN) nuclei express oestrogen receptor (ER)ß and receive afferent projections from osmosensitive neurones that express ERα. However, which subtype of these receptors mediates the effects of oestradiol on vasopressin (AVP) secretion induced by hydromineral challenge has not yet been demonstrated in vivo. Moreover, AVP secretion induced by hyperosmolality is known to involve activation of TRPV1 (transient receptor potential vanilloid, member 1) in magnocellular neurones, although whether oestradiol modulates expression of this receptor is unknown. Thus, the present study aimed to clarify the mechanisms involved in the modulation exerted by oestradiol on AVP secretion, specifically investigating the involvement of ERß, ERα and TRPV1 receptors in response to water deprivation (WD). We observed that treatment with an ERß agonist potentiated AVP secretion and vasopressinergic neuronal activation induced by WD. This increase in AVP secretion induced by WD was reversed by an ERß antagonist. By contrast to ERß, the ERα agonist did not alter plasma AVP concentrations or activation of AVP neurones in the SON and PVN. Additionally, Fos expression in the subfornical organ was not altered by the ERα agonist. TRPV1 mRNA expression was increased by WD in the SON, although this response was not altered by any treatment. The results of the present study suggest that ERß mediates the effects of oestradiol on AVP secretion in response to WD, indicating that the effects of oestradiol occur directly in AVP neurones without affecting TRPV1.

Detection of activity-dependent vasopressin release from neuronal dendrites and axon terminals using sniffer cells.

Our understanding of neuropeptide function within neural networks would be improved by methods allowing dynamic detection of peptide release in living tissue. We examined the usefulness of sniffer cells as biosensors to detect endogenous vasopressin (VP) release in rat hypothalamic slices and from isolated neurohypophyses. Human embryonic kidney cells were transfected to express the human V1a VP receptor (V1aR) and the genetically encoded calcium indicator GCaMP6m. The V1aR couples to Gq11, thus VP binding to this receptor causes an increase in intracellular [Ca2+] that can be detected by a rise in GCaMP6 fluorescence. Dose-response analysis showed that VP sniffer cells report ambient VP levels >10 pM (EC50 = 2.6 nM), and this effect could be inhibited by the V1aR antagonist SR 49059. When placed over a coverslip coated with sniffer cells, electrical stimulation of the neurohypophysis provoked a reversible, reproducible, and dose-dependent increase in VP release using as few as 60 pulses delivered at 3 Hz. Suspended sniffer cells gently plated over a slice adhered to the preparation and allowed visualization of VP release in discrete regions. Electrical stimulation of VP neurons in the suprachiasmatic nucleus caused significant local release as well as VP secretion in distant target sites. Finally, action potentials evoked in a single magnocellular neurosecretory cell in the supraoptic nucleus provoked significant VP release from the somatodendritic compartment of the neuron. These results indicate that sniffer cells can be used for the study of VP secretion from various compartments of neurons in living tissue. NEW & NOTEWORTHY The specific functional roles of neuropeptides in neuronal networks are poorly understood due to the absence of methods allowing their real-time detection in living tissue. Here, we show that cultured "sniffer cells" can be engineered to detect endogenous release of vasopressin as an increase in fluorescence.

Validation of salivary oxytocin and vasopressin as biomarkers in domestic dogs.

BACKGROUND: Oxytocin (OT) and Vasopressin (AVP) are phylogenetically conserved neuropeptides with effects on social behavior, cognition and stress responses. Although OT and AVP are most commonly measured in blood, urine and cerebrospinal fluid (CSF), these approaches present an array of challenges including concerns related to the invasiveness of sample collection, the potential for matrix interference in immunoassays, and whether samples can be collected at precise time points to assess event-linked endocrine responses. NEW METHOD: We validated enzyme-linked immunosorbent assays (ELISAs) for the measurement of salivary OT and AVP in domestic dogs. RESULTS: Both OT and AVP were present in dog saliva and detectable by ELISA and high performance liquid chromatography - mass spectrometry (HPLC-MS). OT concentrations in dog saliva were much higher than those typically detected in humans. OT concentrations in the same samples analyzed with and without sample extraction were highly correlated, but this was not true for AVP. ELISA validation studies revealed good accuracy and parallelism, both with and without solid phase extraction. Collection of salivary samples with different synthetic swabs, or following salivary stimulation or the consumption of food led to variance in results. However, samples collected from the same dogs using different techniques tended to be positively correlated. We detected concurrent elevations in salivary and plasma OT during nursing. COMPARISON WITH EXISTING METHODS: There are currently no other validated methods for measuring OT/AVP in dog saliva. CONCLUSIONS: OT and AVP are present in dog saliva, and ELISAs for their detection are methodologically valid.

Three-dimensional distribution of tyrosine hydroxylase, vasopressin and oxytocin neurones in the transparent postnatal mouse brain.

Over the years, advances in immunohistochemistry techniques have been a critical step in detecting and mapping neuromodulatory substances in the central nervous system. The better quality and specificity of primary antibodies, new staining procedures and the spectacular development of imaging technologies have allowed such progress. Very recently, new methods permitting tissue transparency have been successfully used on brain tissues. In the present study, we combined whole-mount immunostaining for tyrosine hydroxylase (TH), oxytocin (OXT) and arginine vasopressin (AVP), with the iDISCO+ clearing method, light-sheet microscopy and semi-automated counting of three-dimensionally-labelled neurones to obtain a (3D) distribution of these neuronal populations in a 5-day postnatal (P5) mouse brain. Segmentation procedure and 3D reconstruction allowed us, with high resolution, to map TH staining of the various catecholaminergic cell groups and their ascending and descending fibre pathways. We show that TH pathways are present in the whole P5 mouse brain, similar to that observed in the adult rat brain. We also provide new information on the postnatal distribution of OXT and AVP immunoreactive cells in the mouse hypothalamus, and show that, compared to AVP neurones, OXT neurones in the supraoptic (SON) and paraventricular (PVN) nuclei are not yet mature in the early postnatal period. 3D semi-automatic quantitative analysis of the PVN reveals that OXT cell bodies are more numerous than AVP neurones, although their immunoreactive soma have a volume half smaller. More AVP nerve fibres compared to OXT were observed in the PVN and the retrochiasmatic area. In conclusion, the results of the present study demonstrate the utility and the potency of imaging large brain tissues with clearing procedures coupled to novel 3D imaging technologies to study, localise and quantify neurotransmitter substances involved in brain and neuroendocrine functions.

Alcohol Consumption Decreases Oxytocin Neurons in the Anterior Paraventricular Nucleus of the Hypothalamus in Prairie Voles.

BACKGROUND: Alcohol use disorders are associated with dysfunctional social relationships and stress responses. The neuropeptides oxytocin (OT) and vasopressin (AVP) are known to orchestrate or mediate many aspects of social behavior, stress responses, and ingestive behaviors. Because of the overlap between the effects of alcohol and the roles of OT and AVP, we sought to determine whether alcohol consumption altered expression of OT and AVP in the paraventricular nucleus (PVN) of the hypothalamus, one of the key sites for OT and AVP synthesis. METHODS: Pair-housed adult male prairie voles were allowed to consume 15% ethanol versus water in the home cage continuously (Continuous-Access [CA] group) or every other day for 4 hours (Intermittent-Access [IA] group). Control animals never had access to alcohol. After 7 weeks, animals were sacrificed and their brains were removed and immunohistochemical analysis of OT- and AVP-immunopositive neurons was performed. RESULTS: OT-immunopositive neurons were significantly decreased in the anterior PVN in the CA but not IA group, relative to Control animals, suggesting that continuous alcohol consumption decreases the number of OT neurons. There was no effect of alcohol consumption on posterior PVN OT neurons, and no effect on PVN AVP neurons. CONCLUSIONS: These data show that continuous-access voluntary alcohol consumption is associated with decreased OT neurons in the anterior PVN, suggesting that alcohol-induced alterations in the OT system should be investigated as a mechanism for alcohol-related changes in social behavior, stress responses, and exacerbation of alcohol use disorders.

Quantitative mapping reveals age and sex differences in vasopressin, but not oxytocin, immunoreactivity in the rat social behavior neural network.

The neuropeptides vasopressin (AVP) and oxytocin (OT) have been implicated in the regulation of numerous social behaviors in adult and juvenile animals. AVP and OT signaling predominantly occur within a circuit of interconnected brain regions known collectively as the "social behavior neural network" (SBNN). Importantly, AVP and OT signaling within the SBNN has been shown to differentially regulate diverse social behaviors, depending on the age and/or sex of the animal. We hypothesized that variation in the display of these behaviors is due in part to age and sex differences in AVP and OT synthesis within the SBNN. However, a thorough characterization of AVP and OT-immunoreactive (ir) fibers and cell bodies across age and sex within the SBNN has been lacking in rats. We therefore quantified AVP- and OT-ir fibers and cell bodies in 22 subregions of the forebrain SBNN in juvenile and adult, male and female rats. We found numerous age (16 subregions) and sex (10 subregions) differences in AVP-ir fiber fractional areas, and AVP-ir cell body numbers, which were mainly observed in the medial amygdala/bed nucleus of the stria terminalis to lateral septum circuit. In contrast to AVP, we observed no age or sex differences in OT-ir fiber fractional areas or cell bodies in any of the 22 subregions of the forebrain SBNN. Thus, unlike the static pattern observed for OT, AVP innervation of the forebrain SBNN appears to undergo developmental changes, and is highly sexually dimorphic, which likely has significant functional consequences for the regulation of social behavior.

Successful use of closed-loop allostatic neurotechnology for post-traumatic stress symptoms in military personnel: self-reported and autonomic improvements.

BACKGROUND: Military-related post-traumatic stress (PTS) is associated with numerous symptom clusters and diminished autonomic cardiovascular regulation. High-resolution, relational, resonance-based, electroencephalic mirroring (HIRREM®) is a noninvasive, closed-loop, allostatic, acoustic stimulation neurotechnology that produces real-time translation of dominant brain frequencies into audible tones of variable pitch and timing to support the auto-calibration of neural oscillations. We report clinical, autonomic, and functional effects after the use of HIRREM® for symptoms of military-related PTS. METHODS: Eighteen service members or recent veterans (15 active-duty, 3 veterans, most from special operations, 1 female), with a mean age of 40.9 (SD = 6.9) years and symptoms of PTS lasting from 1 to 25 years, undertook 19.5 (SD = 1.1) sessions over 12 days. Inventories for symptoms of PTS (Posttraumatic Stress Disorder Checklist - Military version, PCL-M), insomnia (Insomnia Severity Index, ISI), depression (Center for Epidemiologic Studies Depression Scale, CES-D), and anxiety (Generalized Anxiety Disorder 7-item scale, GAD-7) were collected before (Visit 1, V1), immediately after (Visit 2, V2), and at 1 month (Visit 3, V3), 3 (Visit 4, V4), and 6 (Visit 5, V5) months after intervention completion. Other measures only taken at V1 and V2 included blood pressure and heart rate recordings to analyze heart rate variability (HRV) and baroreflex sensitivity (BRS), functional performance (reaction and grip strength) testing, blood and saliva for biomarkers of stress and inflammation, and blood for epigenetic testing. Paired t-tests, Wilcoxon signed-rank tests, and a repeated-measures ANOVA were performed. RESULTS: Clinically relevant, significant reductions in all symptom scores were observed at V2, with durability through V5. There were significant improvements in multiple measures of HRV and BRS [Standard deviation of the normal beat to normal beat interval (SDNN), root mean square of the successive differences (rMSSD), high frequency (HF), low frequency (LF), and total power, HF alpha, sequence all, and systolic, diastolic and mean arterial pressure] as well as reaction testing. Trends were seen for improved grip strength and a reduction in C-Reactive Protein (CRP), Angiotensin II to Angiotensin 1-7 ratio and Interleukin-10, with no change in DNA n-methylation. There were no dropouts or adverse events reported. CONCLUSIONS: Service members or veterans showed reductions in symptomatology of PTS, insomnia, depressive mood, and anxiety that were durable through 6 months after the use of a closed-loop allostatic neurotechnology for the auto-calibration of neural oscillations. This study is the first to report increased HRV or BRS after the use of an intervention for service members or veterans with PTS. Ongoing investigations are strongly warranted. TRIAL REGISTRATION: NCT03230890 , retrospectively registered July 25, 2017.

Measuring Oxytocin and Vasopressin: Bioassays, Immunoassays and Random Numbers.

In this review, we consider the ways in which vasopressin and oxytocin have been measured since their first discovery. Two different ways of measuring oxytocin in widespread use currently give values in human plasma that differ by two orders of magnitude, and the values measured by these two methods in the same samples show no correlation. The notion that we should accept this seems absurd. Either one (or both) methods is not measuring oxytocin, or, by 'oxytocin', the scientists that use these different methods mean something very different. If these communities are to talk to each other, it is important to validate one method and invalidate the other, or else to establish exactly what each community understands by 'oxytocin'. A similar issue concerns vasopressin: again, different ways of measuring vasopressin give values in human plasma that differ by two orders of magnitude, and it appears that the same explanation for discrepant oxytocin measurements applies to discrepant vasopressin measurements. The first assays for oxytocin and vasopressin measured biological activity directly. When immunoassays were introduced, they encountered problems: high molecular weight factors in raw plasma interfered with the binding of antibodies to the hormones, leading to high and erroneous readings. When these interfering factors were removed by extraction of plasma samples, immunoassays gave measurements consistent with bioassays, with measures of turnover and with the sensitivity of target tissues to exogenous hormone. However, many recent papers use an enzyme-linked immunoassay to measure plasma levels without extracting the samples. Like the first radioimmunassays of unextracted plasma, this generates impossibly high and wholly erroneous measurements.

Peripheral oxytocin and vasopressin: Biomarkers of psychiatric disorders? A comprehensive systematic review and preliminary meta-analysis.

A large array of studies have investigated peripheral oxytocin (OT) and vasopressin (ADH) as potential biomarkers of psychiatric disorders, with highly conflicting and heterogenous findings. We searched Web of KnowledgeSM and Scopus® for English original articles investigating OT and/or ADH levels in different biological fluids (plasma/serum, saliva, urine and cerebrospinal fluid) across several psychiatric disorders. Sixty-four studies were included. We conducted 19 preliminary meta-analyses addressing OT alterations in plasma/serum, saliva, urine and cerebrospinal fluid of 7 psychiatric disorders and ADH alterations in plasma/serum, saliva, urine and cerebrospinal fluid of 6 psychiatric disorders compared to controls. Hedge's g was used as effect size measure, together with heterogeneity analyses, test of publication biases and quality control. None of them (except serum OT in anorexia nervosa) revealed significant differences. There is no convincing evidence that peripheral ADH or OT might be reliable biomarkers in psychiatric disorders. However, the lack of significant results was associated with high methodological heterogeneity, low quality of the studies, small sample size, and scarce reliability of the methods used in previous studies, which need to be validated and standardized.

3D ToF-SIMS Analysis of Peptide Incorporation into MALDI Matrix Crystals with Sub-micrometer Resolution.

The analytical sensitivity in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is largely affected by the specific analyte-matrix interaction, in particular by the possible incorporation of the analytes into crystalline MALDI matrices. Here we used time-of-flight secondary ion mass spectrometry (ToF-SIMS) to visualize the incorporation of three peptides with different hydrophobicities, bradykinin, Substance P, and vasopressin, into two classic MALDI matrices, 2,5-dihydroxybenzoic acid (DHB) and α-cyano-4-hydroxycinnamic acid (HCCA). For depth profiling, an Ar cluster ion beam was used to gradually sputter through the matrix crystals without causing significant degradation of matrix or biomolecules. A pulsed Bi3 ion cluster beam was used to image the lateral analyte distribution in the center of the sputter crater. Using this dual beam technique, the 3D distribution of the analytes and spatial segregation effects within the matrix crystals were imaged with sub-µm resolution. The technique could in the future enable matrix-enhanced (ME)-ToF-SIMS imaging of peptides in tissue slices at ultra-high resolution. Graphical Abstract ᅟ.