Dendritic coding of multiple sensory inputs in single cortical neurons in vivo

Single cortical neurons in the mammalian brain receive signals arising from multiple sensory input channels. Dendritic integration of these afferent signals is critical in determining the amplitude and time course of the neurons' output signals. As of yet, little is known about the spatial and temporal organization of converging sensory inputs. Here, we combined in vivo two-photon imaging with whole-cell recordings in layer 2 neurons of the mouse vibrissal cortex as a means to analyze the spatial pattern of subthreshold dendritic calcium signals evoked by the stimulation of different whiskers. We show that the principle whisker and the surrounding whiskers can evoke dendritic calcium transients in the same neuron. Distance-dependent attenuation of dendritic calcium transients and the corresponding subthreshold depolarization suggest feed-forward activation. We found that stimulation of different whiskers produced multiple calcium hotspots on the same dendrite. Individual hotspots were activated with low probability in a stochastic manner. We show that these hotspots are generated by calcium signals arising in dendritic spines. Some spines were activated uniquely by single whiskers, but many spines were activated by multiple whiskers. These shared spines indicate the existence of presynaptic feeder neurons that integrate and transmit activity arising from multiple whiskers. Despite the dendritic overlap of whisker-specific and shared inputs, different whiskers are represented by a unique set of activation patterns within the dendritic field of each neuron.     

Modeling a layer 4-to-layer 2/3 module of a single column in rat neocortex: interweaving in vitro and in vivo experimental observations

We report a step in constructing an in silico model of a neocortical column, focusing on the synaptic connection between layer 4 (L4) spiny neurons and L2/3 pyramidal cells in rat barrel cortex. It is based first on a detailed morphological and functional characterization of synaptically connected pairs of L4-L2/3 neurons from in vitro recordings and second, on in vivo recordings of voltage responses of L2/3 pyramidal cells to current pulses and to whisker deflection. In vitro data and a detailed compartmental model of L2/3 pyramidal cells enabled us to extract their specific membrane resistivity ( approximately 16,000 ohms x cm(2)) and capacitance ( approximately 0.8 microF/cm(2)) and the spatial distribution of L4-L2/3 synaptic contacts. The average peak conductance per L4 synaptic contact is 0.26 nS for the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and 0.2 nS for NMDA receptors. The in vivo voltage response for current steps was then used to calibrate the model for in vivo conditions in the Down state. Consequently, the effect of a single whisker deflection was modeled by converging, on average, 350 +/- 20 L4 axons onto the modeled L2/3 pyramidal cell. Based on values of synaptic conductance, the spatial distribution of L4 synapses on L2/3 dendrites, and the average in vivo spiking probability of L4 spiny neurons, the model predicts that the feed-forward L4-L2/3 connection on its own does not fire the L2/3 neuron. With a broader distribution in the number of L4 neurons or with slight synchrony among them, the L2/3 model does spike with low probability.     

The inhibitory effect of anandamide on luteinizing hormone-releasing hormone secretion is reversed by estrogen

Because Delta-9-tetrahydrocannabinol (THC) inhibited luteinizing hormone-releasing hormone (LHRH) in male rats, we hypothesized that the endocannabinoid, anandamide (AEA), would act similarly. AEA microinjected intracerebroventricularly (i.c.v.) decreased plasma luteinizing hormone (LH) at 30 min in comparison to values in controls (P < 0.001). The cannabinoid receptor 1 (CB1-r)-specific antagonist, [N-(piperidin-1-yl)-1-(2,4-dichlorophenyl)-5-(4-chlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide] (AM251), produced a significant elevation in plasma LH (P < 0.01). AEA (10(-9) M) decreased LHRH release from medial basal hypothalami incubated in vitro. These results support the concept that endogenous AEA inhibits LHRH followed by decreased LH release in male rats. In ovariectomized (OVX) female rats, AEA i.c.v. also inhibited LH release, but in this case AM251 had an even greater inhibitory effect than AEA. In vitro, AEA had no effect on LHRH in OVX rats. It seems that endogenous AEA inhibits LHRH followed by decreased LH release in OVX rats but that AM251 has an inhibitory action in this case. In striking contrast, in OVX, estrogen-primed (OVX-E) rats, AEA i.c.v. instead of decreasing LH, increased its release. This effect was completely blocked by previous injection of AM251. When medial basal hypothalami of OVX-E rats were incubated, AEA increased LHRH release. The synthesized AEA was higher in OVX-E rats than in OVX and males, indicating that estrogen modifies endocannabinoid levels and effects. The results are interpreted to mean that sex steroids have profound effects to modify the response to AEA. It inhibits LHRH and consequently diminishes LH release in males and OVX females, but stimulates LHRH followed by increased LH release in OVX-E-primed rats.     

The growth speed of microtubules with XMAP215-coated beads coupled to their ends is increased by tensile force

The generation of pulling and pushing forces is one of the important functions of microtubules, which are dynamic and polarized structures. The ends of dynamic microtubules are able to form relatively stable links to cellular structures, so that when a microtubule grows it can exert a pushing force and when it shrinks it can exert a pulling force. Microtubule growth and shrinkage are tightly regulated by microtubule-associated proteins (MAPs) that bind to microtubule ends. Given their localization, MAPs may be exposed to compressive and tensile forces. The effect of such forces on MAP function, however, is poorly understood. Here we show that beads coated with the microtubule polymerizing protein XMAP215, the Xenopus homolog of Dis1 and chTOG, are able to link stably to the plus ends of microtubules, even when the ends are growing or shrinking; at growing ends, the beads increase the polymerization rate. Using optical tweezers, we found that tensile force further increased the microtubule polymerization rate. These results show that physical forces can regulate the activity of MAPs. Furthermore, our results show that XMAP215 can be used as a handle to sense and mechanically manipulate the dynamics of the microtubule tip.     

Resistance of bcr-abl-positive acute lymphoblastic leukemia to daunorubicin is not mediated by mdr1 gene expression

In vitro resistance to anthracyclines is thought to be a poor prognosis in achieving long-term remission in patients with acute lymphoblastic leukemia (ALL). Expression of a multidrug resistance gene (mdr1) that codes for 170 Kd transmembrane glycoprotein is responsible for conferring resistance to malignant cells to anthracyclines. The t(9:22) translocation, resulting in bcr-abl fusion gene, is commonly found in B-lineage ALL and is known to be a poor prognostic factor for long-term remission. To investigate whether resistance to anthracyclines contributes to poor prognosis in bcr-abl-positive ALL, we studied daunorubicin sensitivity by an in vitro colorimetric methyl tetrazolium (MTT) assay in B-lineage ALL patients who were bcr-abl-positive and compared them with the B-lineage, age-matched bcr-abl-negative group. We also looked for and compared the presence of mdr1 gene expression in these two groups of patients by RT-PCR. Of the 46 patients included in the study, 16 (34.7%) were positive for the bcr-abl fusion gene. mdr1 gene expression was seen in 14 of these 46 patients (30.4%). However, the expression of the mdr1 gene was relatively lower in the bcr-abl-positive group (3 out of 16, 18.7%) compared to the bcr-abl-negative group (11 out of 30, 36.6%). The median LD(50) of daunorubicin (concentration lethal to 50% of the leukemic blasts) differed significantly between bcr-abl-positive and -negative patients (P = 0.018). This in vitro study suggests that bcr-abl-positive ALL is relatively resistant to daunorubicin, but this resistance is not mediated through mdr1 gene expression.     

Enhanced insulin-stimulated glycogen synthesis in response to insulin, metformin or rosiglitazone is associated with increased mRNA expression of GLUT4 and peroxisomal proliferator activator receptor gamma co-activator 1

Aims/hypothesis The aim of this study was to determine the effect of several antidiabetic agents on insulin-stimulated glycogen synthesis, as well as on mRNA expression.Methods Cultured primary human skeletal myotubes obtained from six healthy subjects were treated for 4 or 8 days without or with glucose (25 mmol/l), insulin (400 pmol/l), rosiglitazone (10 mol/l), metformin (20 mol/l) or the AMP-activated kinase activator 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) (200 mol/l). After this, insulin-stimulated glycogen synthesis was determined. mRNA levels of the glucose transporters GLUT1 and GLUT4, the peroxisomal proliferator activator receptor gamma (PPAR gamma) co-activator 1 (PGC1) and the myocyte-specific enhancer factors (MEF2), MEF2A, MEF2C and MEF2D were determined using real-time PCR analysis after 8 days exposure to the various antidiabetic agents.Results Insulin-stimulated glycogen synthesis was significantly increased in cultured human myotubes treated with insulin, rosiglitazone or metformin for 8 days, compared with non-treated cells. Furthermore, an 8-day exposure of myotubes to 25 mmol/l glucose impaired insulin-stimulated glycogen synthesis. In contrast, treatment with AICAR was without effect on insulin-mediated glycogen synthesis. Exposure to insulin, rosiglitazone or metformin increased mRNA expression of PGC1 and GLUT4, while AICAR or 25 mmol/l glucose treatment increased GLUT1 mRNA expression. Metformin also increased mRNA expression of the MEF2 isoforms.Conclusions/interpretation Enhanced insulin-stimulated glycogen synthesis in human skeletal muscle cell culture coincides with increased GLUT4 and PGC1 mRNA expression following treatment with various antidiabetic agents. These data show that chronic treatment of human myotubes with insulin, metformin or rosiglitazone has a direct positive effect on insulin action and mRNA expression.     

Low frequency of myeloid progenitor cells in chronic idiopathic neutropenia of adults may be related to increased production of TGF-beta1 by bone marrow stromal cells.

Previous studies in our laboratory have shown that patients with chronic idiopathic neutropenia of adults (CINA) have increased serum levels of inflammatory cytokines including IL-1beta. Since IL-1beta may affect bone marrow stromal cell function, a study was designed to investigate the capacity of patients' stromal cells to produce adequate amounts of haemopoietic growth factors or excessive amounts of inhibitors of myelopoiesis in long-term bone marrow cultures (LTBMCs). The study was carried out on 52 CINA patients and 19 normal controls. We found that CINA patients had significantly low numbers of marrow lineage-specific CD34+ cells, including CFU-GM and CD34+/CD33+ cells. Stromal cells from patients' LTBMCs failed to stimulate CFU-GM colony formation by normal marrow cells in a manner comparable to that of stromal cells of controls. Patients' LTBMC supernatants had normal or increased amounts of G-CSF. Detectable amounts of supernatant GM-CSF were found in 35% of patients and 19% of controls. IL-3 and MIP-1alpha were not detected in any supernatant fluid. Moreover, supernatants from patients' LTBMCs had increased concentrations of IL-6 and TGF-beta1, which strongly correlated with serum IL-1beta. About 82% of our patients had TGF-beta1 values higher than the upper limit of values found in the controls. Individual TGF-beta1 values inversely correlated with the number of circulating neutrophils and the frequency of marrow CD34+/CD33+ cells. We suggest that increased levels of serum IL-1beta, resulting from an underlying low-grade chronic inflammatory process, may stimulate marrow stromal cells to produce both haemopoietic growth factors and inhibitors of myelopoiesis. Since steady-state myelopoiesis results from a balance between negative- and positive-acting cytokines, it seems very probable that the increased production of TGF-beta1 by bone marrow microenvironment in CINA patients may suppress myelopoiesis and contribute, to some extent, to the pathogenesis of neutropenia in affected subjects.     

Down-regulation of transforming growth factor β1/activin receptor-like kinase 1 pathway gene expression by herbal compound 861 is related to deactivation of LX-2 cells

AIM: To investigate the effect of herbal compound 861(Cpd861) on the transforming growth factor-131 (TGFβ1)/activin receptor-like kinase 1 (ALK1, type I receptor)signaling-pathway-related gene expression in the LX-2 cell line, and the inhibitory mechanism of Cpd861 on the activation of LX-2 cells.METHODS: LX-2 cells were treated with TGFβ1(5 ng/mL) Cpd861 (0.1 mg/mL), TGFβ1 (5 ng/mL) plus Cpd861 (5 ng/mL) for 24 h to investigate the effect of Cpd861 on the TGFβ1/ALK1 pathway. Real-time PCR was performed to examine the expression of α-SMA (α-smooth muscle actin), ALK1, Id1 (inhibitor of differentiation 1). Western blotting was carried out to measure the levels of α-SMA and phosphorylated Smadl,and immunocytochemical analysis for the expression of α-SMA.RESULTS: In LX-2 cells, TGFβ1/ALK1-pathway-related gene expression could be stimulated by TGFβ1, which led to excessive activation of the cells. Cpd861 decreased the activation of LX-2 cells by reducing the expression of α-SMA mRNA and protein expression. This effect was related to inhibition of the above TGFβ1/ALK1-pathwayrelated expression of genes such as Id1 and ALK1, and phosphorylation of Smad1 in LX-2 cells, even with TGFβ1 co-treatment for 24 h.CONCLUSION: Cpd861 can restrain the activation of LX-2 cells by inhibiting the TGFβ1/ALK1/Smad1 pathway.     

Down-regulation of transforming growth factor β1/activin receptor-like kinase 1 pathway gene expression by herbal compound 861 is related to deactivation of LX-2 cells

AIM： To investigate the effect of herbal compound 861 （Cpd861） on the transforming growth factor-β1 （TGFβ1）/ activin receptor-like kinase 1 （ALK1, type Ⅰ receptor） signaling-pathway-related gene expression in the LX-2 cell line, and the inhibitory mechanism of Cpd861 on the activation of LX-2 cells.
METHODS： LX-2 cells were treated with TGFβ1 （5 ng/mL） Cpd861 （0.1 mg/mL）, TGFβ1 （5 ng/mL） plus Cpd861 （5 ng/mL） for 24 h to investigate the effect of Cpd861 on the TGFβ1/ALK1 pathway. Real-time PCR was performed to examine the expression of α-SMA （α-smooth muscle actin）, ALK1, Id1 （inhibitor of differentiation 1）. Western blotting was carried out to measure the levels of α-SMA and phosphorylated Smad1, and immunocytochemical analysis for the expression of α-SMA.
RESULTS： In LX-2 cells, TGFβ1/ALK1-pathway-related gene expression could be stimulated by TGFβ1, which led to excessive activation of the cells. Cpd861 decreased the activation of LX-2 cells by reducing the expression of α-SMA mRNA and protein expression. This effect was related to inhibition of the above TGFβ1/ALK1-pathway- related expression of genes such as Id1 and ALK1, and phosphorylation of Smad1 in LX-2 cells, even with TGFβ1 co-treatment for 24 h.
CONCLUSION： Cpd861 can restrain the activation of LX-2 cells by inhibiting the TGFβ1/ALK1/Smad1 pathway.     

Local release of human neutrophil lipocalin (HNL), IL-8, and TNF-alpha is decreased as response to topical prednisolone treatment in distal ulcerative colitis and proctitis

The local release of human neutrophil lipocalin, considered to be highly specific for neutrophil granulocyte activation, and interleukin-8 and tumor necrosis factor- were studied in 11 patients with distal ulcerative colitis and proctitis before and during treatment with steroid enemas. A rectal perfusion technique for sampling and specific immunoassays for analysis were used. In responders (N = 8) the concentrations of all proteins decreased during the study. There was a close correlation between human neutrophil lipocalin concentrations and treatment response. Tumor necrosis factor- showed an initial decline in concentrations irrespective of treatment outcome and preceded the decline of human neutrophil lipocalin and interleukin-8. We conclude that decreased neutrophil degranulation is correlated with treatment outcome. Furthermore, an important role of tumor necrosis factor- in the process of stimulating neutrophil activation and degranulation in ulcerative colitis is suggested.     

Whole inactivated virus influenza vaccine is superior to subunit vaccine in inducing immune responses and secretion of proinflammatory cytokines by DCs

Background For protection against (re‐)infection by influenza virus not only the magnitude of the immune response but also its quality in terms of antibody subclass and T helper profile is important. Information about the type of immune response elicited by vaccination is therefore urgently needed. Objectives The aim of the study was to evaluate in detail the immune response elicited by three current influenza vaccine formulations and to shed light on vaccine characteristics which determine this response. Methods Mice were immunized with whole inactivated virus (WIV), virosomes (VS) or subunit vaccine (SU). Following subsequent infection with live virus, serum antibody titers and Th cell responses were measured. The effects of the vaccines on cytokine production by conventional and plasmacytoid dendritic cells were investigated in vitro. Results and conclusions In Balb/c mice (Th2 prone) as well as in C57Bl/6 mice (Th1 prone), WIV induced consistently higher hemagglutination‐inhibition titers and virus‐neutralizing antibody titers than VS or SU. In contrast to VS and SU, WIV stimulated the production of the antibody subclasses IgG2a (Balb/c) and IgG2c (C57BL/6), considered to be particularly important for viral clearance, and activation of IFN‐γ‐producing T cells. Similar to live virus, WIV stimulated the production of proinflammatory cytokines by conventional dendritic cells and IFN‐α by plasmacytoid cells, while VS and SU had little effect on cytokine synthesis by either cell type. We conclude that vaccination with WIV in contrast to VS or SU results in the desired Th1 response presumably by induction of type I interferon and other proinflammatory cytokines.     

Insulin resistance is accompanied by increased von Willebrand factor levels in nondiabetic women: a study of offspring of type 2 diabetic subjects compared to offspring of nondiabetic subjects

OBJECTIVES: To examine whether levels of von Willebrand factor (vWF), fibrinogen and fibronectin are related to a parental history of type 2 diabetes and to determine possible explanatory factors for high versus low vWF and fibrinogen. DESIGN: Cross-sectional study. SUBJECTS, MAIN OUTCOME MEASURES: We compared vWF, fibrinogen and fibronectin in 88 nondiabetic offspring of type 2 diabetic subjects (relatives) and 103 offspring of nondiabetic subjects (controls). Other measurements included urinary albumin excretion rate, blood pressure, lipid profile and insulin resistance using homeostasis model assessment (HOMAIR). RESULTS: There were no significant differences in vWF (1.12 vs. 1.06 IU x mL(-1), P = 0.296), fibrinogen (3.2 vs. 3.1 g x L(-1); P = 0.263) or fibronectin (0.39 vs. 0.40 g x L(-1), P = 0.448) between relatives and controls. With multiple logistic regression we determined explanatory factors for high versus low vWF and fibrinogen. Age (P < 0.01), urinary albumin excretion rate (P < 0.05), ischaemic heart disease (IHD) (P < 0.05) were found to be significant explanatory factors for vWF above the median (1.10 IU x mL(-1)). Interaction between insulin resistance and sex was found. Odds ratio for high versus low insulin resistance was 18.39 (P < 0.001) for women and 1.92 (P = 0.32) for men. Body mass index (BMI) (P < 0.05), sex (P < 0.01), smoking status (P < 0.05) and IHD (P < 0.01) were significant explanatory factors for fibrinogen above the median (3.1 g x L(-1)). CONCLUSIONS: Levels of vWF, fibrinogen and fibronectin were not influenced by a parental history of type 2 diabetes. Insulin resistance was found to be a significant risk indicator for high vWF only in women. This may indicate that insulin resistance is a higher risk factor for women than for men, when the outcome is endothelial dysfunction possibly resulting in overt cardiovascular disease.     

Cholecystokinin secretion is suppressed by glucagon-like peptide-1: clue to the mechanism of the adverse gallbladder events of GLP-1-derived drugs

Abstract

Objective: Recent randomized and controlled trials of drugs derived from the gut hormone glucagon-like peptide-1 (GLP-1) show that the most frequent adverse symptoms are gastrointestinal, including gallbladder-related side effects such as cholithiasis and cholecystitis. Since the gut hormone cholecystokinin (CCK) stimulates bile secretion and regulates gallbladder motility and emptying, we examined the effect of GLP-1 on the secretion of CCK in normal subjects and patients with type 1 diabetes mellitus.

Materials and methods: Plasma was sampled from 10 healthy subjects and 10 patients with diabetes. With plasma glucose concentrations clamped between 6 and 9?nmol/l, GLP-1 or saline was infused for 240?min during and after a meal. The plasma concentrations of CCK were measured with a highly specific radioimmunoassay.

Results: Basal plasma concentrations of CCK were similar in the normal subjects and in the diabetes patients. During the meal, the CCK concentrations rose significantly during saline infusion, whereas the GLP-1 infusion suppressed the secretion of CCK significantly in both normal subjects and in the diabetes patients.

Conclusions: The results show that GLP-1 suppresses the secretion of CCK after a meal in normal and diabetic subjects. The suppression attenuates the gallbladder contractility. Our data, therefore, offer an explanation for the increased risk of adverse gallbladder events during treatment with GLP-1-derived drugs.     

Effective lysis of model thrombi by a t-PA mutant (A473S) that is resistant to alpha2-antiplasmin

This study used two mutants of tissue-type plasminogen activator (t-PA) with resistance to inhibitors of fibrinolysis to define the contribution of plasminogen activator inhibitor (PAI)-1 and alpha2-antiplasmin (alpha2-AP) to the control of fibrin lysis. Wild-type t-PA was compared with KHRR296-299AAAA, which is resistant to PAI-1, and with A473S, which is resistant to alpha2-AP. We examined these forms of t-PA in model systems that are physiologically relevant. Neutralization of alpha2-AP was essential for lysis of plasma clots, irrespective of their platelet content, by either wild-type t-PA or KHRR296-299AAAA. In marked contrast, A473S lysed plasma clots without neutralization of alpha2-AP. Model thrombi, with structures similar to in vivo thrombi, were lysed slowly by wild-type t-PA; the rate and extent of lysis were enhanced by the addition of antibodies to alpha2-AP or PAI-1. A473S was more effective than wild-type t-PA without the addition of antibodies by virtue of its resistance to alpha2-AP. This resistance was remarkable, in that no complex formed between A473S t-PA and alpha2-AP, even after extended incubation, when 50% of wild-type t-PA could be converted to complex. Comparison of A473S and KHRR296-299AAAA mutants showed their similar effectiveness in lysis of platelet-rich model thrombi. Thus, PAI-1 and alpha2-AP contribute approximately equally to the inhibition of thrombus lysis. This study underlines the functional significance of alpha2-AP as a direct inhibitor of t-PA and further explains the basis of the accepted role of alpha2-AP as a regulator of fibrin persistence and thrombus resistance to lysis.