Synaptic plasticity regulation mediated by BDNF: functional and morphological study

2010/2011The long-term potentiation (LTP) represents a widely studied form of synaptic plasticity related to learning and memory processes, which involves a long-lasting strengthening of synaptic connections through changes of their transmission and cytoarchitecture. The induction of LTP is classically achieved by tetanic stimulation of presynaptic components but it is also possible to in- duce chemically a long-term potentiation of the synaptic efficacy, thus enhancing a larger number of synapses compared to electrical stimulation and facilitating the biochemical and morphological study. The first part of this thesis is a methodological study of glycine and tetraethylammonium (TEA) induced chemical LTP (cLTP) in cultured hippocampal cells. Brief glycine (in Mg2+-free) application activate NMDA receptors, whereas TEA blocks of K+ channels inducing a depolariza- tion responsible for Ca2+ influx. Both drugs were briefly superfused and mEPSCs were monitored for all the duration of the experiments (≃60 min). This was considered as a necessary step to detect later the role of the Brain Derived Neurophic Factor (BDNF) in cLTP. Healthy hippocampal cells were dissected from rats of postnatal day 1-2. After a period of 10-12 days in vitro the cells reached optimal density, a typical mature pyramidal neuron morphology, and an extended dendritic arborization which facilitates synaptic contacts. At this stage patch-clamp technique in the whole-cell configuration was used to study the electrophysiological properties of pyramidal hippocampal neurons, able to produce spontaneous electrical activity. cLTP was tested recording miniature excitatory postsynaptic currents (mEPSCs) in voltage-clamp mode focusing on changes in their amplitude and frequency. A significant decrease in mEPSCs inter-event intervals was observed after glycine and TEA application, without significant changes in aptitudes. Therefore 20 min after glycine application an increase (≃ 61.6 %) in mEPSCs frequency was observed. A similar result was obtained also after TEA application (≃ 66 %). Following cLTP we observed also morphological changes such as an increase in density and a remodeling of different classes of dendritic spines. The role of BDNF in this cLTP model was assessed testing by ELISA assay the total BDNF expres- sion on cell lysate and by blocking Tropomyosin Receptor Kinase (Trk) with K252A. A significant increase in BDNF levels (≃ 120 %) was observed 50 min after cLTP induction. A switch from cLTP into cLTD was observed blocking Trk receptors. Moreover, confocal images collected before and after chemical potentiation in the presence of K252A showed a significant reduction (≃10%) in the average spine density both at the proximal and distal level. A significant reduction of the p-TrkB/TrkB ratio, after both gly- and TEA-LTP, was observed in distal dendrites compared to the soma. This therefore suggests a translocation of the activated receptor from periphery to the soma.XXIV Ciclo198

The Emerging Role of the Autophagy Process in Children with Celiac Disease: Current Status and Research Perspectives

Celiac disease (CD) affects approximately 1% of the population in Europe and North America, but the number of patients currently undiagnosed is estimated to be far higher than that of diagnosed cases owing to the presence of prevalent forms with nonspecific symptoms. The toxicity of gliadin in children with CD is not destroyed through digestion with gastropancreatic enzymes. An innate immunity to gliadin plays a key role in the development of CD. Autophagy, a physiological catabolic process, plays also a crucial role in the pathogenesis of several inflammatory diseases. Recent studies have described functional involvement of the regulation of autophagy within a pediatric CD cohort. Furthermore, the contribution of autophagy has been highlighted in the degradation and in the reduction of extracellular release of gliadin peptides, thus suggesting novel molecular targets to counteract gliadin-induced toxicity in CD

Firing properties of genetically identified dorsal raphe serotonergic neurons in brain slices

Tonic spiking of serotonergic neurons establishes serotonin levels in the brain. Since the first observations, slow regular spiking has been considered as a defining feature of serotonergic neurons. Recent studies, however, have revealed the heterogeneity of serotonergic neurons at multiple levels, comprising their electrophysiological properties, suggesting the existence of functionally distinct cellular subpopulations. In order to examine in an unbiased manner whether serotonergic neurons of the dorsal raphe nucleus are heterogeneous, we used a non-invasive loose-seal cell-attached method to record α1 adrenergic receptor-stimulated spiking of a large sample of neurons in brain slices obtained from transgenic mice lines that express fluorescent marker proteins under the control of serotonergic system-specific Tph2 and Pet-1 promoters. We found wide homogeneous distribution of firing rates, well fitted by a single Gaussian function (r2 = 0.93) and independent of anatomical location (P = 0.45), suggesting that in terms of intrinsic firing properties, serotonergic neurons in the dorsal raphe nucleus represent a single cellular population. Characterization of the population in terms of spiking regularity was hindered by its dependence on the firing rate. For instance, the coefficient of variation of the interspike intervals, a common measure of spiking irregularity, is of limited usefulness since it correlates negatively with the firing rate (r = -0.33, P < 0.0001). Nevertheless, the majority of neurons exhibited regular, pacemaker-like activity, with coefficient of variance of the interspike intervals lower than 0.5 in ~97% of cases. Unexpectedly, a small percentage of neurons (~1%) exhibited a particular spiking pattern, characterized by low frequency (~0.02 - 0.1 Hz) oscillations in the firing rate. Transitions between regular and oscillatory firing were observed, suggesting that the oscillatory firing is an alternative firing pattern of serotonergic neurons

Management of Celiac Patients with Growth Failure

Celiac disease (CD) may be considered as a systemic immune-mediated disorder that is triggered by dietary gluten in genetically susceptible subjects. CD children and adolescents show typical intestinal symptoms such as diarrhea, loss of weight and abdominal distension, or extraintestinal signs, the so-called nonclassical CD, such as short stature and delayed puberty. An endocrinological investigation including an evaluation of growth hormone (GH) secretion should be performed in CD subjects who show no catch-up growth after at least 1 year on a strict gluten-free diet (GFD) in the presence of a seronegativity of anti-transglutaminase and/or antiendomysial antibodies. When the diagnosis of GH deficiency is formulated, a substitutive therapy with GH must be promptly started to obtain a complete catch-up growth. The long-term effects of GH therapy in CD children who follow a strict GFD are comparable to those found in children with idiopathic GHD. A widely documented association has been observed between CD and type I diabetes mellitus and/or Hashimoto thyroiditis and/or Addison’s disease. During follow-up, pediatricians should check antibody serology, thyroid and adrenal function and glucose-metabolic profile in order to verify the compliance with both diet and GH treatment. Adherence to a strict gluten-free diet promotes regular linear growth and may prevent CD complications as well as the onset of other autoimmune diseases

Role of chronic exsposure to cigarette smoke on endoglin/CD105 expression in airway epithelium.

Dysregulation of airway epithelial cell function related to cigarette smoke exposure plays an important role in the pathophysiology of COPD and is associated to tissue damage and disease severity. CD105 is a component of the receptor complex of TGF-β, a pleiotropic cytokine involved in cellular proliferation, differentiation and migration. CD105 regulates the expression of different components of the extracellular matrix suggesting a role of CD105 in cellular transmigration and remodeling processes. The aim of the present study was to investigate the expression of endoglin/CD105 in airway epithelium of COPD patients and its involvement in tissue remodeling and progression of COPD. We evaluated the immunoreactivity for CD105 expression in bronchial biopsies isolated from COPD patients and healthy controls (HC). The analysis of metaplastic epithelium was performed in bronchial biopsies by Image Analysis software (Leica Quantimet system). Finally, we investigated the expression of CD105 protein receptor in human bronchial epithelial cells (16HBE cells) exposed to 5% Cigarette Smoke Extract (CSE) for 12 days by western blot. We found that the CD105 immunoreactivity was significantly higher in bronchial epithelium of COPD than HC. Morphometric analysis of bioptic samples of COPD showed an increase of the immunoreactivity for CD105 in the area of metaplastic than in not metaplastic epithelium. Long term exposure to CSE significantly up-regulated CD105 expression in 16HBE. Chronic inflammation due to cigarette smoke might play a critic role on the alteration of CD105 protein expression in COPD, promoting tissue remodeling, angiogenesis and dysregulation of physiological reparative mechanisms, leading to squamous metaplasia

Dual-beam confocal light-sheet microscopy via flexible acousto-optic deflector

Confocal detection in digital scanned laser light-sheet fluorescence microscopy (DSLM) has been established as a gold standard method to improve image quality. The selective line detection of a complementary metal-oxide-semiconductor camera (CMOS) working in rolling shutter mode allows the rejection of out-of-focus and scattered light, thus reducing background signal during image formation. Most modern CMOS have two rolling shutters, but usually only a single illuminating beam is used, halving the maximum obtainable frame rate. We report on the capability to recover the full image acquisition rate via dual confocal DSLM by using an acousto-optic deflector. Such a simple solution enables us to independently generate, control and synchronize two beams with the two rolling slits on the camera. We show that the doubling of the imaging speed does not affect the confocal detection high contrast

Impact of safety-related dose reductions or discontinuations on sustained virologic response in HCV-infected patients: Results from the GUARD-C Cohort

BACKGROUND: Despite the introduction of direct-acting antiviral agents for chronic hepatitis C virus (HCV) infection, peginterferon alfa/ribavirin remains relevant in many resource-constrained settings. The non-randomized GUARD-C cohort investigated baseline predictors of safety-related dose reductions or discontinuations (sr-RD) and their impact on sustained virologic response (SVR) in patients receiving peginterferon alfa/ribavirin in routine practice. METHODS: A total of 3181 HCV-mono-infected treatment-naive patients were assigned to 24 or 48 weeks of peginterferon alfa/ribavirin by their physician. Patients were categorized by time-to-first sr-RD (Week 4/12). Detailed analyses of the impact of sr-RD on SVR24 (HCV RNA <50 IU/mL) were conducted in 951 Caucasian, noncirrhotic genotype (G)1 patients assigned to peginterferon alfa-2a/ribavirin for 48 weeks. The probability of SVR24 was identified by a baseline scoring system (range: 0-9 points) on which scores of 5 to 9 and <5 represent high and low probability of SVR24, respectively. RESULTS: SVR24 rates were 46.1% (754/1634), 77.1% (279/362), 68.0% (514/756), and 51.3% (203/396), respectively, in G1, 2, 3, and 4 patients. Overall, 16.9% and 21.8% patients experienced 651 sr-RD for peginterferon alfa and ribavirin, respectively. Among Caucasian noncirrhotic G1 patients: female sex, lower body mass index, pre-existing cardiovascular/pulmonary disease, and low hematological indices were prognostic factors of sr-RD; SVR24 was lower in patients with 651 vs. no sr-RD by Week 4 (37.9% vs. 54.4%; P = 0.0046) and Week 12 (41.7% vs. 55.3%; P = 0.0016); sr-RD by Week 4/12 significantly reduced SVR24 in patients with scores <5 but not 655. CONCLUSIONS: In conclusion, sr-RD to peginterferon alfa-2a/ribavirin significantly impacts on SVR24 rates in treatment-naive G1 noncirrhotic Caucasian patients. Baseline characteristics can help select patients with a high probability of SVR24 and a low probability of sr-RD with peginterferon alfa-2a/ribavirin

Serotonin Deficiency Increases Context-Dependent Fear Learning Through Modulation of Hippocampal Activity

Brain serotonin (5-hydroxytryptamine, 5-HT) system dysfunction is implicated in exaggerated fear responses triggering various anxiety-, stress-, and trauma-related disorders. However, the underlying mechanisms are not well understood. Here, we investigated the impact of constitutively inactivated 5-HT synthesis on context-dependent fear learning and extinction using tryptophan hydroxylase 2 ( ) knockout mice. Fear conditioning and context-dependent fear memory extinction paradigms were combined with c-Fos imaging and electrophysiological recordings in the dorsal hippocampus (dHip). mutant mice, completely devoid of 5-HT synthesis in brain, displayed accelerated fear memory formation and increased locomotor responses to foot shock. Furthermore, recall of context-dependent fear memory was increased. The behavioral responses were associated with increased c-Fos expression in the dHip and resistance to foot shock-induced impairment of hippocampal long-term potentiation (LTP). In conclusion, increased context-dependent fear memory resulting from brain 5-HT deficiency involves dysfunction of the hippocampal circuitry controlling contextual representation of fear-related behavioral responses.</p

Serotonin Deficiency Increases Context-Dependent Fear Learning Through Modulation of Hippocampal Activity

Brain serotonin (5-hydroxytryptamine, 5-HT) system dysfunction is implicated in exaggerated fear responses triggering various anxiety-, stress-, and trauma-related disorders. However, the underlying mechanisms are not well understood. Here, we investigated the impact of constitutively inactivated 5-HT synthesis on context-dependent fear learning and extinction using tryptophan hydroxylase 2 (Tph2) knockout mice. Fear conditioning and context-dependent fear memory extinction paradigms were combined with c-Fos imaging and electrophysiological recordings in the dorsal hippocampus (dHip). Tph2 mutant mice, completely devoid of 5-HT synthesis in brain, displayed accelerated fear memory formation and increased locomotor responses to foot shock. Furthermore, recall of context-dependent fear memory was increased. The behavioral responses were associated with increased c-Fos expression in the dHip and resistance to foot shock-induced impairment of hippocampal long-term potentiation (LTP). In conclusion, increased context-dependent fear memory resulting from brain 5-HT deficiency involves dysfunction of the hippocampal circuitry controlling contextual representation of fear-related behavioral responses