Behavior Stability and Individual Differences in Pavlovian Extended Conditioning.

How stable and general is behavior once maximum learning is reached? To answer this question and understand post-acquisition behavior and its related individual differences, we propose a psychological principle that naturally extends associative models of Pavlovian conditioning to a dynamical oscillatory model where subjects have a greater memory capacity than usually postulated, but with greater forecast uncertainty. This results in a greater resistance to learning in the first few sessions followed by an over-optimal response peak and a sequence of progressively damped response oscillations. We detected the first peak and trough of the new learning curve in our data, but their dispersion was too large to also check the presence of oscillations with smaller amplitude. We ran an unusually long experiment with 32 rats over 3,960 trials, where we excluded habituation and other well-known phenomena as sources of variability in the subjects' performance. Using the data of this and another Pavlovian experiment by Harris et al. (2015), as an illustration of the principle we tested the theory against the basic associative single-cue Rescorla-Wagner (RW) model. We found evidence that the RW model is the best non-linear regression to data only for a minority of the subjects, while its dynamical extension can explain the almost totality of data with strong to very strong evidence. Finally, an analysis of short-scale fluctuations of individual responses showed that they are described by random white noise, in contrast with the colored-noise findings in human performance.

Inhibition of MicroRNA-155 Supports Endothelial Tight Junction Integrity Following Oxygen-Glucose Deprivation.

BACKGROUND: Brain microvascular endothelial cells form a highly selective blood brain barrier regulated by the endothelial tight junctions. Cerebral ischemia selectively targets tight junction protein complexes, which leads to significant damage to cerebral microvasculature. Short noncoding molecules called microRNAs are implicated in the regulation of various pathological states, including endothelial barrier dysfunction. In the present study, we investigated the influence of microRNA-155 (miR-155) on the barrier characteristics of human primary brain microvascular endothelial cells (HBMECs). METHODS AND RESULTS: Oxygen-glucose deprivation was used as an in vitro model of ischemic stroke. HBMECs were subjected to 3 hours of oxygen-glucose deprivation, followed by transfections with miR-155 inhibitor, mimic, or appropriate control oligonucleotides. Intact normoxia control HBMECs and 4 oxygen-glucose deprivation-treated groups of cells transfected with appropriate nucleotide were subjected to endothelial monolayer electrical resistance and permeability assays, cell viability assay, assessment of NO and human cytokine/chemokine release, immunofluorescence microscopy, Western blot, and polymerase chain reaction analyses. Assessment of endothelial resistance and permeability demonstrated that miR-155 inhibition improved HBMECs monolayer integrity. In addition, miR-155 inhibition significantly increased the levels of major tight junction proteins claudin-1 and zonula occludens protein-1, while its overexpression reduced these levels. Immunoprecipitation and colocalization analyses detected that miR-155 inhibition supported the association between zonula occludens protein-1 and claudin-1 and their stabilization at the HBMEC membrane. Luciferase reporter assay verified that claudin-1 is directly targeted by miR-155. CONCLUSIONS: Based on these results, we conclude that miR-155 inhibition-induced strengthening of endothelial tight junctions after oxygen-glucose deprivation is mediated via its direct target protein claudin-1.

In vivo inhibition of miR-155 significantly alters post-stroke inflammatory response.

BACKGROUND: MicroRNA miR-155 is implicated in modulation of the inflammatory processes in various pathological conditions. In our previous studies, we demonstrated that in vivo inhibition of miR-155 promotes functional recovery after mouse experimental stroke. In the present study, we explored if this beneficial effect is associated with miR-155 inhibition-induced alterations in post-stroke inflammatory response. METHODS: Intravenous injections of a specific miR-155 inhibitor were initiated at 48 h after mouse distal middle cerebral artery occlusion (dMCAO). Temporal changes in the expression of cytokines and key molecules associated with cytokine signaling were assessed at 7, 14, and 21 days after dMCAO, using mouse cytokine gene and protein arrays and Western blot analyses. Electron and immunofluorescence confocal microscopy techniques were used to evaluate the ultrastructural changes, as well as altered expression of specific phenotypic markers, at different time points after dMCAO. RESULTS: In the inhibitor-injected mice (inhibitor group), there was a significant decrease in CCL12 and CXCL3 cytokine expression at 7 days and significantly increased levels of major cytokines IL-10, IL-4, IL-6, MIP-1α, IL-5, and IL-17 at 14 days after dMCAO. These temporal changes correlated with altered expression of miR-155 target proteins SOCS-1, SHIP-1, and C/EBP-ß and phosphorylation levels of cytokine signaling regulator STAT-3. Electron microscopy showed decreased number of phagocytically active peri-vascular microglia/macrophages in the inhibitor samples. Immunofluorescence and Western blot of these samples demonstrated that expression of leukocyte/ macrophage marker CD45 and phagocytosis marker CD68 was reduced at 7 days, and in contrast, significantly increased at 14 days after dMCAO, as compared to controls. CONCLUSIONS: Based on our findings, we propose that in vivo miR-155 inhibition following mouse stroke significantly alters the time course of the expression of major cytokines and inflammation-associated molecules, which could influence inflammation process and tissue repair after experimental cerebral ischemia.

In Vivo Inhibition of miR-155 Promotes Recovery after Experimental Mouse Stroke.

A multifunctional microRNA, miR-155, has been recently recognized as an important modulator of numerous biological processes. In our previous in vitro studies, miR-155 was identified as a potential regulator of the endothelial morphogenesis. The present study demonstrates that in vivo inhibition of miR-155 supports cerebral vasculature after experimental stroke. Intravenous injections of a specific miR-155 inhibitor were initiated at 48 h after mouse distal middle cerebral artery occlusion (dMCAO). Microvasculature in peri-infarct area, infarct size, and animal functional recovery were assessed at 1, 2, and 3 weeks after dMCAO. Using in vivo two-photon microscopy, we detected improved blood flow and microvascular integrity in the peri-infarct area of miR-155 inhibitor-injected mice. Electron microscopy revealed that, in contrast to the control group, these animals demonstrated well preserved capillary tight junctions (TJs). Western blot analysis data indicate that improved TJ integrity in the inhibitor-injected animals could be associated with stabilization of the TJ protein ZO-1 and mediated by the miR-155 target protein Rheb. MRI analysis showed significant (34%) reduction of infarct size in miR-155 inhibitor-injected animals at 21 d after dMCAO. Reduced brain injury was confirmed by electron microscopy demonstrating decreased neuronal damage in the peri-infarct area of stroke. Preservation of brain tissue was reflected in efficient functional recovery of inhibitor-injected animals. Based on our findings, we propose that in vivo miR-155 inhibition after ischemia supports brain microvasculature, reduces brain tissue damage, and improves the animal functional recovery. Significance statement: In the present study, we investigated an effect of the in vivo inhibition of a microRNA, miR-155, on brain recovery after experimental cerebral ischemia. To our knowledge, this is the first report describing the efficiency of intravenous anti-miRNA injections in a mouse model of ischemic stroke. The role of miRNAs in poststroke revascularization has been unexplored and in vivo regulation of miRNAs during the subacute phase of stroke has not yet been proposed. Our investigation introduces a new and unexplored approach to cerebral regeneration: regulation of poststroke angiogenesis and recovery through direct modulation of specific miRNA activity. We expect that our findings will lead to the development of novel strategies for regulating neurorestorative processes in the postischemic brain.

Insulin hypersecretion in islets from diet-induced hyperinsulinemic obese female mice is associated with several functional adaptations in individual ß-cells.

Insulin resistance and hyperinsulinemia are generally associated with obesity. Obese nondiabetic individuals develop a compensatory ß-cell response to adjust insulin levels to the increased demand, maintaining euglycemia. Although several studies indicate that this compensation relies on structural changes, the existence of ß-cell functional adaptations is incompletely understood. Here, we fed female mice with a high-fat diet (HFD) for 12 weeks. These animals became obese, hyperinsulinemic, insulin-resistant, and mildly glucose-intolerant while fed, and fasting glycemia was comparable in HFD and control mice. Islets from HFD animals exhibited increased ß-cell mass and hypertrophy. Additionally, they had enhanced insulin gene expression and content and augmented glucose-induced insulin secretion. Electrophysiological examination of ß-cells from both groups showed no differences in KATP channel open probability and conductance. However, action potentials elicited by glucose had larger amplitude in obese mice. Glucose-induced Ca²âº signals in intact islets, in isolated ß-cells, and individual ß-cells within islets were also increased in HFD mice. Additionally, a higher proportion of glucose-responsive cells was present in obese mice. In contrast, whole-cell Ca²âº current densities were similar in both groups. Capacitance measurements showed that depolarization-evoked exocytosis was enhanced in HFD ß-cells compared with controls. Although this augment was not significant when capacitance increases of the whole ß-cell population were normalized to cell size, the exocytotic output varied significantly when ß-cells were distributed by size ranges. All these findings indicate that ß-cell functional adaptations are present in the islet compensatory response to obesity.

Role of leptin in the pancreatic ß-cell: effects and signaling pathways.

Leptin plays an important role in the control of food intake, energy expenditure, metabolism, and body weight. This hormone also has a key function in the regulation of glucose homeostasis. Although leptin acts through central and peripheral mechanisms to modulate glucose metabolism, the pancreatic ß-cell of the endocrine pancreas is a critical target of leptin actions. Leptin receptors are present in the ß-cell, and their activation directly inhibits insulin secretion from these endocrine cells. The effects of leptin on insulin occur also in the long term, since this hormone inhibits insulin gene expression as well. Additionally, ß-cell mass can be affected by leptin through changes in proliferation, apoptosis, or cell size. All these different functions in the ß-cell are triggered by leptin as a result of the large diversity of signaling pathways that this hormone is able to activate in the endocrine pancreas. Therefore, leptin can participate in glucose homeostasis owing to different levels of modulation of the pancreatic ß-cell population. Furthermore, it has been proposed that alterations in this level of regulation could contribute to the impairment of ß-cell function in obesity states. In the present review, we will discuss all these issues with special emphasis on the effects and pathways of leptin signaling in the pancreatic ß-cell.

The clock gene Rev-erbα regulates pancreatic ß-cell function: modulation by leptin and high-fat diet.

Disturbances of circadian rhythms have been associated with obesity and type 2 diabetes. The nuclear receptor Rev-erbα was suggested to link circadian rhythms and metabolism in peripheral tissues. The aim of the present study was to dissect the role of this clock gene in the pancreatic ß-cell function and to analyze whether its expression is modulated by leptin and diet-induced obesity. To address the function of Rev-erbα, we used small interfering RNA in mouse islet cells and in MIN-6 cells. Cell proliferation was measured by bromodeoxyuridine incorporation, apoptosis by the terminal deoxynucleotidyl transferase dUTP nick end labeling technique, insulin secretion by RIA, and gene expression by RT-PCR. Pancreatic islets were isolated at different zeitgeber times 0, 6, and 12 after 6 wk of high-fat diet treatment, and then gene expression and insulin secretion were determined. Rev-erbα down-regulation by small interfering RNA treatment in islet cells and MIN-6 cells impaired glucose-induced insulin secretion, decreased the expression of key lipogenic genes, and inhibited ß-cell proliferation. In vivo and in vitro leptin treatment increased Rev-erbα expression in isolated islets through a MAPK pathway. High-fat diet treatment disrupted the circadian Rev-erbα gene expression profile along with insulin secretion, indicating an important role of this clock gene in ß-cell function. These results indicate that the clock gene Rev-erbα plays multiple functions in the pancreatic ß-cell. Although the increase in Rev-erbα expression may promote ß-cell adaptation in different metabolic situations, its deregulation may lead to altered ß-cell function.

Monocytes from cystic fibrosis patients are locked in an LPS tolerance state: down-regulation of TREM-1 as putative underlying mechanism.

Cystic Fibrosis (CF) is an inherited pleiotropic disease that results from abnormalities in the gene that codes for the chloride channel, Cystic Fibrosis Transmembrane Conductance Regulator (CFTR). CF patients are frequently colonized by several pathogens, but the mechanisms that allow colonization in spite of apparently functional immune systems are incompletely understood. In this paper we show that blood peripheral monocytes isolated from CF patients are found in an endotoxin tolerance state, yet this is not due to a deficient TLR activation. On the other hand, levels of the amplifier of inflammatory responses, TREM-1 (Triggering Receptor Expressed on Myeloid cells), are notably down-regulated in monocytes from patients, in comparison to those extracted from healthy volunteers. Furthermore, the soluble form of TREM-1 (sTREM-1) was not detected in the sera of patients. Additionally, and in strict contrast to patients who suffer from Chronic Obstructive Pulmonary Disease (COPD), CF monocytes challenged ex vivo with LPS neither up-regulated membrane-anchored TREM-1 nor sTREM-1. Finally, similar levels of PGE(2) expression and p65 translocation into the nucleus were found in both patients and healthy volunteers, thus suggesting that TREM-1 regulation is neither controlled by PGE(2) levels nor by p65 activation in this case. However, PU.1 translocation into the nucleus was significantly higher in CF monocytes than in controls, suggesting a role for this transcription factor in the control of TREM-1 expression. We conclude that down-regulation of TREM-1 expression in cystic fibrosis patients is at least partly responsible for the endotoxin tolerance state in which their monocytes are locked.