Glutamic acid decarboxylase and glutamate receptor changes during tolerance and dependence to benzodiazepines

Protracted administration of diazepam elicits tolerance, whereas discontinuation of treatment results in signs of dependence. Tolerance to the anticonvulsant action of diazepam is present in an early phase (6, 24, and 36 h) but disappears in a late phase (72–96 h) of withdrawal. In contrast, signs of dependence such as decrease in open-arm entries on an elevated plus-maze and increased susceptibility to pentylenetetrazol-induced seizures were apparent 96 h (but not 12, 24, or 48 h) after diazepam withdrawal. During the first 72 h of withdrawal, tolerance is associated with changes in the expression of GABAA (γ-aminobutyric acid type A) receptor subunits (decrease in γ2 and α1; increase in α5) and with an increase of mRNA expression of the most abundant form of glutamic acid decarboxylase (GAD), GAD67. In contrast, dl-α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor GluR1 subunit mRNA and cognate protein, which are normal during the early phase of diazepam withdrawal, increase by approximately 30% in cortex and hippocampus in association with the appearance of signs of dependence 96 h after diazepam withdrawal. Immunohistochemical studies of GluR1 subunit expression with gold-immunolabeling technique reveal that the increase of GluR1 subunit protein is localized to layer V pyramidal neurons and their apical dendrites in the cortex, and to pyramidal neurons and in their dendritic fields in hippocampus. The results suggest an involvement of GABA-mediated processes in the development and maintenance of tolerance to diazepam, whereas excitatory amino acid-related processes (presumably via AMPA receptors) may be involved in the expression of signs of dependence after withdrawal.

γ-Aminobutyric acid type A receptors modulate cAMP-mediated long-term potentiation and long-term depression at monosynaptic CA3–CA1 synapses

cAMP induces a protein-synthesis-dependent late phase of long-term potentiation (LTP) at CA3–CA1 synapses in acute hippocampal slices. Herein we report cAMP-mediated LTP and long-term depression (LTD) at monosynaptic CA3–CA1 cell pairs in organotypic hippocampal slice cultures. After bath application of the membrane-permeable cAMP analog adenosine 3′,5′-cyclic monophosphorothioate, Sp isomer (Sp-cAMPS), synaptic transmission was enhanced for at least 2 h. Consistent with previous findings, the late phase of LTP requires activation of cAMP-dependent protein kinase A and protein synthesis. There is also an early phase of LTP induced by cAMP; the early phase depends on protein kinase A but, in contrast to the later phase, does not require protein synthesis. In addition, the cAMP-induced LTP is associated with a reduction of paired-pulse facilitation, suggesting that presynaptic modification may be involved. Furthermore, we found that Sp-cAMPS induced LTD in slices pretreated with picrotoxin, a γ-aminobutyric acid type A (GABAA) receptor antagonist. This form of LTD depends on protein synthesis and protein phosphatase(s) and is accompanied by an increased ratio of failed synaptic transmission. These results suggest that GABAA receptors can modulate the effect of cAMP on synaptic transmission and thus determine the direction of synaptic plasticity.

Dynamics of Immature Secretory Granules: Role of Cytoskeletal Elements during Transport, Cortical Restriction, and F-Actin-dependent Tethering

Secretory granules store neuropeptides and hormones and exhibit regulated exocytosis upon appropriate cellular stimulation. They are generated in the trans-Golgi network as immature secretory granules, short-lived vesicular intermediates, which undergo a complex and poorly understood maturation process. Due to their short half-life and low abundance, real-time studies of immature secretory granules have not been previously possible. We describe here a pulse/chase-like system based on the expression of a human chromogranin B-GFP fusion protein in neuroendocrine PC12 cells, which permits direct visualization of the budding of immature secretory granules and their dynamics during maturation. Live cell imaging revealed that newly formed immature secretory granules are transported in a direct and microtubule-dependent manner within a few seconds to the cell periphery. Our data suggest that the cooperative action of microtubules and actin filaments restricts immature secretory granules to the F-actin-rich cell cortex, where they move randomly and mature completely within a few hours. During this maturation period, secretory granules segregate into pools of different motility. In a late phase of maturation, 60% of secretory granules were found to be immobile and about half of these underwent F-actin-dependent tethering.

Impaired hippocampal plasticity in mice lacking the Cbeta1 catalytic subunit of cAMP-dependent protein kinase.

Neural pathways within the hippocampus undergo use-dependent changes in synaptic efficacy, and these changes are mediated by a number of signaling mechanisms, including cAMP-dependent protein kinase (PKA). The PKA holoenzyme is composed of regulatory and catalytic (C) subunits, both of which exist as multiple isoforms. There are two C subunit genes in mice, Calpha and Cbeta, and the Cbeta gene gives rise to several splice variants that are specifically expressed in discrete regions of the brain. We have used homologous recombination in embryonic stem cells to introduce an inactivating mutation into the mouse Cbeta gene, specifically targeting the Cbeta1-subunit isoform. Homozygous mutants showed normal viability and no obvious pathological defects, despite a complete lack of Cbeta1. The mice were analyzed in electrophysiological paradigms to test the role of this isoform in long-term modulation of synaptic transmission in the Schaffer collateral-CA1 pathway of the hippocampus. A high-frequency stimulus produced potentiation in both wild-type and Cbeta1-/- mice, but the mutants were unable to maintain the potentiated response, resulting in a late phase of long-term potentiation that was only 30% of controls. Paired pulse facilitation was unaffected in the mutant mice. Low-frequency stimulation produced long-term depression and depotentiation in wild-type mice but failed to produce lasting synaptic depression in the Cbeta1 -/- mutants. These data provide direct genetic evidence that PKA, and more specifically the Cbeta1 isoform, is required for long-term depression and depotentiation, as well as the late phase of long-term potentiation in the Schaffer collateral-CA1 pathway.

Hippocampal long-term depression and depotentiation are defective in mice carrying a targeted disruption of the gene encoding the RI beta subunit of cAMP-dependent protein kinase.

The cAMP-dependent protein kinase (PKA) has been shown to play an important role in long-term potentiation (LTP) in the hippocampus, but little is known about the function of PKA in long-term depression (LTD). We have combined pharmacologic and genetic approaches to demonstrate that PKA activity is required for both homosynaptic LTD and depotentiation and that a specific neuronal isoform of type I regulatory subunit (RI beta) is essential. Mice carrying a null mutation in the gene encoding RI beta were established by use of gene targeting in embryonic stem cells. Hippocampal slices from mutant mice show a severe deficit in LTD and depotentiation at the Schaffer collateral-CA1 synapse. This defect is also evident at the lateral perforant path-dentate granule cell synapse in RI beta mutant mice. Despite a compensatory increase in the related RI alpha protein and a lack of detectable changes in total PKA activity, the hippocampal function in these mice is not rescued, suggesting a unique role for RI beta. Since the late phase of CA1 LTP also requires PKA but is normal in RI beta mutant mice, our data further suggest that different forms of synaptic plasticity are likely to employ different combinations of regulatory and catalytic subunits.

Magnetic anisotropy reveals Neogene tectonic overprint in highly strained carbonate mylonites from the Morcles nappe, Switzerland

The anisotropy of magnetic susceptibility (AMS) has been measured with low- and high-field methods, in deformed carbonate rocks along the Morcles nappe shear zone (Helvetic Alps). High-field measurements at room temperature and 77 K enable the separation of the ferrimagnetic, paramagnetic and diamagnetic anisotropy. The ferrimagnetic sub-fabric is generally insignificant in these rocks, contributing less than 10% to the total AMS. AMS results for both the separated diamagnetic and paramagnetic subfabrics are consistent with the regional shear movement in the late-stage formation of the Helvetic nappes, as seen in the Morcles nappe, whose inverted limb indicate shear displacement towards the northwest. The diamagnetic anisotropy correlates well quantitatively with the calculated magnetic anisotropy based on the calcite texture. There is a gradational change in the degree of anisotropy related to the strain gradient along the shear zone. A more complex magnetic fabric, resulting from partial overprinting due to displacement along the Simplon–Rhône fault, is evident at one site near the root zone of the nappe. Partial overprinting of the magnetic fabric appears to have taken place in two locations farther up the shear zone as well. This late phase deformation is associated with recent exhumation of the Mont Blanc and Belledonne external massifs and orogen parallel extension, and is reflected by the AMS. Rocks with bulk susceptibility ∼0 SI, and simple mineral compositions are ideal for low temperature high-field torque, as this method helps to enhance the paramagnetic susceptibility and anisotropy, which may otherwise be masked by the mixed magnetic contributions of the composite magnetic fabric.

Characterization of the glial scar tissue in a murine model of spinal cord compression, with focus on hyaluronan

Spinal cord injury (SCI) is a devastating neurological disorder that affects thousands of people each year. Although in recent decades significant progress has been made in relation to understanding the molecular and cellular events underlying the nervous damage, spinal cord injury is still a highly disabling condition for which there is no curative therapy. People affected by spinal cord injuries manifested dysfunction or loss, temporary or permanent, of motor, sensory and / or autonomic functions depending on the spinal lesion damaged. Currently, the incidence rate of this type of injury is approximately 15-40 cases per million people worldwide. At the origin of these lesions are: road accidents, falls, interpersonal violence and the practice of sports. In this work we placed the hypothesis that HA is one of the component of the scar tissue formed after a compressive SCI, that it is likely synthetised by the perilesional glial cells and that it might support the permeation of the glial scar during the late phase of SCI. Nowadays, much focus is drawn on the recovery of CNS function, made impossible after SCI due to the high content of sulfated proteoglycans in the extracellular matrix. Counterbalancing the ratio between these proteoglycans and hyaluronic acid could be one of the experimental therapy to re-permeate the glial scar tissue formed after SCI, making possible axonal regrowth and functional recovery. Therefore, we established a model of spinal cord compression in mice and studied the glial scar tissue, particularly through the characterization of the expression of enzymes related to the metabolism of HA and the subsequent concentration thereof at different distances of the lesion epicenter. Our results show that the lesion induced in mice shows results similar to those produced in human lesions, in terms of histologic similarities and behavioral results. but these animals demonstrate an impressive spontaneous reorganization mechanism of the spinal cord tissue that occurs after injury and allows for partial recovery of the functions of the CNS. As regards the study of the glial scar, changes were recorded at the level of mRNA expression of enzymes metabolizing HA i.e., after injury there was a decreased expression of HA synthases 1-2 (HAS 1-2) and an increase of the expression HAS3 synthase mRNA, as well as the enzymes responsible for the HA catabolism, HYAL 1-2. But the amount of HA measured through the ELISA test was found unchanged after injury, it is not possible to explain this fact only with the change of expression of enzymes. At two weeks and in response to SCI, we found synthesized HA by reactive astrocytes and probably by others like microglial cells as it was advanced by the HA/GFAP+ and HA/IBA1+ cells co-location.

Frühdiagenetische Karbonatzementation in glazigenen und fluviatilen Sedimenten des Wesertales NW Hameln

In the Weser valley NW Hameln early diagenetic carbonate cementation is present in the upper Elsterian to Drenthe sediments. Examination of the calcite cement indicated a multiphase formation. Cementation occured mainly in vadose environment above ground-water table. This process generated predominantly brown calcite cement with a crystal size of <5 to 20pm. Next to meniscus-cement this fine-crystalline calcite forms further cements typical for vadose zone. In one layer of the „Mittelterrasse'-sediments, deposited in the late Elsterian age, a calcite cement is present, which may already have formed in the Holstein Interglacial. Cementation started in the upper-most layers of the „Mittelterrasse'-sediments in a late phase of „Mittelterrasse“-accumulation in this area. The so formed consolidated layers were deformed by the Drentheglacier. After ice melting cementation continued in at least 3 phases, creating nonluminiscent phreatic, orange to red luminiscent vadose and nonluminiscent vadose calcite cement.

Investigating the role of target cell availability in the pathogenesis of feline immunodeficiency virus infection

Feline immunodeficiency virus (FIV) is a naturally occurring lentivirus of domestic cats, which shares many similarities with its human counterpart, human immunodeficiency virus (HIV). FIV infects its main target cell, the CD4+ T lymphocyte, via interactions with its primary receptor CD134 (an activation marker expressed on activated CD4+ T lymphocytes), and, the chemokine receptor CXCR4. According to the different ways in which FIV isolates interact with CD134, FIV may be categorised into two groups. The first group contains strains that tend to dominate during the earlier phase of infection, such as GL8 and CPG41. These strains are characterized by their requirement for an additional interaction with the second cysteine rich domain (CRD2) of the CD134 molecule and are classified as “CRD2-dependent” strains. The second group, on the other hand, contains either laboratory-adapted isolates or isolates that emerge after several years of infection, such as PPR or the GL8 variants that emerged in cats 6 years post experimental infection and were studied in this thesis. These isolates are designated “CRD2-independent” as they can infect target cells without interacting with CRD2 of the CD134 molecule. This study provides the first evidence that FIV compartmentalisation is related to FIV-CD134 usage and the tissue availability of CD134+ target cells. In tissue compartments containing high levels of CD134+ cells such as peripheral blood and lymph nodes, CRD2-dependent viruses predominated, whereas CRD2-independent viruses predominated in compartments with fewer CD134+ cells, such as the thymus. The dynamics of CD4+CD134+ T lymphocytes at different stages of FIV infection were also described. The levels of CD4+CD134+ T lymphocytes, which were very high in the early phase, gradually decreased in the later phase of infection. The dynamics of CD4+CD134+ T lymphocyte numbers appeared to correlate with FIV tropism switching, as more CRD2-independent viruses were isolated from cats in the late phase of infection. Moreover, it was observed that pseudotypes bearing Envs of CRD2-dependent variants infected CD134+ target cells more efficiently than pseudotypes bearing Envs of CRD2-independent variants, confirming the selective advantage of CRD2-dependent variants in environments with high levels of CD134+ target cells. In conclusion, this study demonstrated that target cell types and numbers, as well as their dynamics, play important roles in the selection and expansion of FIV variants within the viral quasispecies. Improved understanding of the roles of target cells in FIV transmission and pathogenesis will provide important information required for the development of an improved, more successful protective FIV vaccine and will provide insight into the development of effective vaccines against other lentiviral infections such as HIV.

Anti-nociceptive and anti-inflammatory potentials of fractions from the extract of Tetracarpidium conophorum leaf in rats and mice

Tetracarpidium conophorum (TC) (Euphorbiaceae) is a perennial woody climbing shrub in low bush forest of some parts of West Africa and used among the natives for relief of ailments accompanying pain and inflammation. In this study, the analgesic and anti-inflammatory effects of the methanolic extract (METC) and fractions (ethyl acetate, F1 and n-hexane, F2) of Tetracarpidium conophorum leaf were evaluated in rat and mice. The analgesic activity was evaluated using acetic acid-induced writhing, formalin-induced paw licking and hot plate test models. Carrageenan-induced paw oedema was used to assess anti-inflammatory activity in rats. The mechanism of action of (TC) was explored by the use of naloxone, a non-selective opioid receptor blocker. The highest analgesic effect was observed in F2 extract at 57.21% inhibition and was further studied on various analgesic and anti-inflammatory models in graded doses. F2 significantly inhibited the late phase of formalin-induced paw licking and prolong hot plate latency at 55±1°C. The n-hexane fraction also significantly inhibited carrageenan-induced paw oedema in rats at 100 and 200mg/kg doses significantly (p< 0.001) and reduced paw licking response by 85.08% compared with control. Naloxone, an opioid receptor antagonist, did not significantly affect the changes observed with n-hexane fraction, thus ruling out the possibility of the involvement of opioid receptors in the analgesic actions of Tetracarpidium conophorum. Phytochemical screening showed that the leaf extracts contain alkaloids, tannins, saponins and cardenolides. The investigations showed that Tetracarpidium conophorum possesses significant anti-nociceptive and anti-inflammatory activities that should be explored.

Lo sviluppo del movimento sabbatiano in Italia. Binyamin ben El'azar Coen Vitale da Reggio e il suo 'Et ha-zamir (עת הזמיר, Il tempo del canto)

La mia tesi si concentra sullo sviluppo del movimento sabbatiano in Italia tra il XVII e il XVIII secolo, una fase tarda del Sabbatianesimo. Il tentativo è quello di descrivere questo post-sabbatianesimo e i suoi rappresentanti attraverso lo studio di figure come quelle di Binyamin ben El‘azar Coen Vitale da Reggio (1651-1730), un rabbino piemontese identificato da Gershom Scholem - il padre degli studi sabbatiani - come un modello nel panorama sabbatiano italiano. Dopo una contestualizzazione storica e culturale che compara il mondo cristiano e quello ebraico dell'epoca, e il misticismo ebraico italiano con quello originario di Safed in Galilea, ho analizzato la produzione letteraria di Binyamin Coen. Mi sono concentrata sui suoi testi a stampa, traducendo integralmente e analizzando la versione a stampa di‘Et ha-zamir, "Il tempo del canto", una raccolta di poesie cabalistiche (piyyutim), composta da poesie per i giorni della settimana e per le festività principali. la prim a edizione a stampa del "Tempo del canto" risale al 1707, e fu stampata a Venezia. Il testo era utilizzato nel contesto delle confraternite cabalistiche dei ghetti italiani, e spicca per l'uso molto particolare della metrica legata alla cabbala. ‘Et ha-zamir è infatti perfettamente inserito in un'epoca che vide una vasta diffusione della mistica ebraica.

Acute symptoms, not rectally administered sucralfate, predict for late radiation proctitis: Longer term follow-up of a phase III trial - trans-Tasman radiation oncology group

Image : To assess the potential for sucralfate administered rectally to reduce the risk of late rectal morbidity in patients undergoing nonconformal radiotherapy (RT) for carcinoma of the prostate and to study the variables potentially contributing to late rectal morbidity and particularly to explore the relationship between acute and late toxicity. Image : Eighty-six patients with localized prostate carcinoma were randomized in a double-blind, placebo-controlled study to a daily enema of 3 g of sucralfate in a 15-mL suspension or the same suspension without sucralfate. The enema began the first day of RT and was continued for 2 weeks after treatment completion. The primary end point of the study was acute Radiation Therapy Oncology Group (RTOG)/European Organization for Research and Treatment of Cancer (EORTC) toxicity; however, the patients were followed for an additional 5 years on a 6-month basis. The evaluation included late RTOG/EORTC toxicity and a patient self-assessment questionnaire. Image : With a median follow-up of 5 years, the Kaplan-Meier probability of late Grade 2 RTOG/EORTC toxicity was 12% (95% confidence interval [CI] 2–22%) for placebo and 5% (95% CI 0–12%) for sucralfate (p = 0.26). The probability of late rectal bleeding was 59% (95% CI 45–73%) for placebo and 54% (95% CI 40–68%) for sucralfate. No statistically significant difference was found between the treatment arms for the peak incidence of any of the other patient self-assessment variables. Cox proportional hazards modeling indicated acute RTOG/EORTC toxicity of Grade 2 or greater was associated with a hazard ratio of 2.74 (95% CI 1.31–5.73) for the development of late toxicity of Grade 1 or greater. Substituting the patient self-assessment variables for acute RTOG/EORTC toxicity revealed that rectal pain of a moderate or severe grade during RT was the best predictor of the subsequent development of late toxicity, with a hazard ratio of 3.44 (95% CI 1.68–7). Image : The results of this study do not support the use of sucralfate administered rectally as a method for reducing the late toxicity of nonconformal RT for prostate cancer. There appears to be an association between the development of acute and subsequent late toxicity, although the nature of this association remains to be determined

Schistosoma mansoni granuloma in late evolutive phase, in a case of tumoral form in man

INTRODUCTION: Authors describe human schistosomal granuloma in late chronic phase, from the morphological and evolutionary viewpoints. METHODS: The study was based on a histological analysis of two fragments obtained from a surgical biopsy of peritoneum and large intestine of a 42-year-old patient, with a pseudotumoral form mimicking a peritoneal carcinomatosis associated to the schistosomiasis hepatointestinal form. RESULTS: Two hundred and three granulomas were identified in the pseudotumor and 27 in the intestinal biopsy, with similar morphological features, most in the late chronic phase, in fibrotic healing. A new structural classification was suggested for granulomas: zone 1 (internal), 2 (intermediate) and 3 (external). CONCLUSIONS: Regarding granuloma as a whole, we may conclude that fibrosis is likely to be controlled by different and independent mechanisms in the three zones of the granuloma. Lamellar fibrosis in zone 3 seems to be controlled by matrix mesenchymal cells (fibroblasts and myoepithelial cells) and by inflammatory exudate cells (lymphocytes, plasmocytes, neutrophils, eosinophils). Annular fibrosis in zone 2, comprising a dense fibrous connective tissue, with few cells in the advanced phase, would be controlled by epithelioid cells involving zone 1 in recent granulomas. In zone 1, replacing periovular necrosis, an initialy loose and tracery connective neoformation, housing stellate cells or with fusiform nuclei, a dense paucicellular nodular connctive tissue emerges, probably induced by fibroblasts. In several granulomas, one of the zones is missing and granuloma is represented by two of them: Z3 and Z2, Z3 and Z1 or Z2 and Z1 and, ultimately, by a scar.

Late Mogollon communities: four sites of the Tularosa phase, western New Mexico / [by] Paul S. Martin, John B. Rinaldo [and] Eloise R. Barter.

v.49:no.1(1957)

Evaluation of Long-Term Outcomes, Cytogenetic and Molecular Responses with Imatinib Mesylate in Early and Late Chronic-Phase Chronic Myeloid Leukemia: A Report from a Single Institute

Here we compare the management and survival outcomes of chronic myeloid leukemia (CML) patients who had early or late imatinib mesylate (IM) therapy. The cytogenetic and molecular responses of 189 CML patients were analyzed. Of this group, 121 patients were classified as the early chronic phase (ECP) group and started IM within 12 months of diagnosis. The other 68 patients were classified as the late chronic phase (LCP) group who had been treated with interferon (IFN)-alpha-2 and crossed over to IM more than 12 months after diagnosis. The overall rates of complete cytogenetic response (CCyR) and major molecular response (MMR) at last follow-up were 83.6 and 78.1% in the ECP and LCP groups, respectively. The CCyR rates were 89.3 (for ECP patients) versus 73.5% (for LCP patients; p < 0.0001). At last follow-up, 82.4% ECP and 64.2% LCP patients had achieved an MMR (p < 0.0001). No significant differences were noted between the two groups with regard to survival outcomes. Our experience reveals that IM is an effective rescue therapy in most CML LCP patients who are intolerant or in whom IFN-alpha therapy fails. Such therapeutic options should be considered in LCP patients, particularly in countries where IM may not be available. Copyright (C) 2012 S. Karger AG, Basel