Maternal deprivation causes CaMKII downregulation and modulates glutamate, norepinephrine and serotonin in limbic brain areas in a rat model of single prolonged stress.

BACKGROUND: Early life stress is a major risk factor for later development of psychiatric disorders, including post-traumatic stress disorder (PTSD). An intricate relationship exists between various neurotransmitters (such as glutamate, norepinephrine or serotonin), calcium/calmodulin-dependent protein kinase II (CaMKII), as an important regulator of glutamatergic synaptic function, and PTSD. Here, we developed a double-hit model to investigate the interaction of maternal deprivation (MD) as an early life stress model and single prolonged stress (SPS) as a PTSD model at the behavioral and molecular levels. METHODS: Male Wistar rats exposed to these stress paradigms were subjected to a comprehensive behavioral analysis. In hippocampal synaptosomes we investigated neurotransmitter release and glutamate concentration. The expression of CaMKII and the content of monoamines were determined in selected brain regions. Brain-derived neurotrophic factor (BDNF) mRNA was quantified by radioactive in situ hybridization. RESULTS: We report a distinct behavioral phenotype in the double-hit group. Double-hit and SPS groups had decreased hippocampal presynaptic glutamatergic function. In hippocampus, double-hit stress caused a decrease in autophosphorylation of CaMKII. In prefrontal cortex, both SPS and double-hit stress had a similar effect on CaMKII autophosphorylation. Double-hit stress, rather than SPS, affected the norepinephrine and serotonin levels in prefrontal cortex, and suppressed BDNF gene expression in prefrontal cortex and hippocampus. LIMITATIONS: The study was conducted in male rats only. The affected brain regions cannot be restricted to hippocampus, prefrontal cortex and amygdala. CONCLUSION: Double-hit stress caused more pronounced and distinct behavioral, molecular and functional changes, compared to MD or SPS alone.

Alpha-gal syndrome-Food or drug allergy: A case report.

Alpha-gal syndrome is an immunoglobulin E-mediated hypersensitivity characterized by delayed allergic reactions to ingested products containing alpha-gal carbohydrate. We present a patient with recurrent urticaria and suspected repaglinide hypersensitivity, who was eventually diagnosed with alpha-gal syndrome, wanting to emphasize possible drug allergy misdiagnosis and required caution with the medication choice.

Impaired migratory phenotype of CD4+ T cells in Parkinson's disease.

Dysfunctions in the immune system appear implicated in both disease onset and progression of Parkinson's disease (PD). Neurodegeneration observed in the brain of PD patients has been associated with neuroinflammation that is linked to alterations in peripheral adaptive immunity, where CD4+ T cells are key players. In the present study, we elucidated the immunological aspect of PD by employing a wide range of cellular assays, immunocytochemistry and flow cytometry to examine CD4+ T cells. We particularly investigated the role of CD4+ T cell migration in the proper functioning of the adaptive immune system. Our data reveal the altered migration potential of CD4+ T cells derived from PD patients, along with impaired mitochondrial positioning within the cell and reduced mitochondrial functionality. In addition, a cross-sectional study of p11 levels in CD4+ T cell subsets showed a differentially increased level of p11 in Th1, Th2 and Th17 populations. Taken together, these results demonstrate major impairments in the functionality of peripheral CD4+ T cells in PD.

Adolescent and young adult allergic asthma treatment challenges.

The transition from paediatric care to adult care is often difficult, especially in children with chronic diseases like asthma. A significant number of children reach remission throughout puberty; consequently, they are not tracked down for subsequent follow-ups and are not included in transition programmes to adult care. This case report focuses on a young adult with asthma that began in childhood and went into remission during adolescence, only to experience a recurrence when the patient was a young adult. Due to failing to complete the transition process into adult care services, she had poor adherence to therapy and asthma control.Adherence and asthma control significantly improved after a multidisciplinary approach in an adult care setting. In conclusion, appropriate transition and a multidisciplinary approach are critical for the effective management of asthma in young adults.

α-Synuclein induced cholesterol lowering increases tonic and reduces depolarization-evoked synaptic vesicle recycling and glutamate release.

α-Synuclein (α-syn) is a key molecule linked to Parkinson's disease pathology. Physiologically, the monomeric α-syn in the presynaptic termini is involved in regulation of neurotransmission, but the pathophysiology of extracellular monomeric α-syn is still unknown. Utilizing both in vivo and in vitro approaches, we investigated how extracellular α-syn impact presynaptic structure and function. Our data revealed that treatment with exogenous α-syn leads to increased tonic and decreased depolarization-evoked synaptic vesicle (SV) recycling and glutamate release. This was associated with mobilization of molecularly distinct SV pools and reorganization of active zone components. Our study also showed that exogenous α-syn impaired neuronal cholesterol level and that the cholesterol binding domain of α-syn was sufficient to exert the same presynaptic phenotype as the full-length protein. The present study sheds new light on physiological functions of extracellular α-syn in overall maintenance of presynaptic activity that involves the reorganization of both presynaptic compartment and cholesterol-rich plasma membrane domains.

Unexpected cause of dyspnoea in a patient with allergic rhinitis.

A defect in one part of the immune system may affect the whole system. As a result, there may be a myriad of immunological diseases, which are often masked with the one disease that has the most prominent symptoms. This case report presents a patient with long-lasting allergic rhinitis who recently developed dyspnoea in exertion with suspected asthma development. After extensive diagnostic processing, asthma was dismissed, and diagnosis of selective IgA deficiency and coeliac disease with consequential iron deficiency anaemia was established. The patient was treated with parenteral iron and a gluten-free diet, which corrected her anaemia and led to the disappearance of dyspnoea. This paper aims to show the interplay between different immunological disorders and the possible causal connection between them.

Ketamine decreases neuronally released glutamate via retrograde stimulation of presynaptic adenosine A1 receptors.

Ketamine produces a rapid antidepressant response in patients with major depressive disorder (MDD), but the underlying mechanisms appear multifaceted. One hypothesis, proposes that by antagonizing NMDA receptors on GABAergic interneurons, ketamine disinhibits afferens to glutamatergic principal neurons and increases extracellular glutamate levels. However, ketamine seems also to reduce rapid glutamate release at some synapses. Therefore, clinical studies in MDD patients have stressed the need to identify mechanisms whereby ketamine decreases presynaptic activity and glutamate release. In the present study, the effect of ketamine and its antidepressant metabolite, (2R,6R)-HNK, on neuronally derived glutamate release was examined in rodents. We used FAST methodology to measure depolarization-evoked extracellular glutamate levels in vivo in freely moving or anesthetized animals, synaptosomes to detect synaptic recycling ex vivo and primary cortical neurons to perform functional imaging and to examine intracellular signaling in vitro. In all these versatile approaches, ketamine and (2R,6R)-HNK reduced glutamate release in a manner which could be blocked by AMPA receptor antagonism. Antagonism of adenosine A1 receptors, which are almost exclusively expressed at nerve terminals, also counteracted ketamine's effect on glutamate release and presynaptic activity. Signal transduction studies in primary neuronal cultures demonstrated that ketamine reduced P-T286-CamKII and P-S9-Synapsin, which correlated with decreased synaptic vesicle recycling. Moreover, systemic administration of A1R antagonist counteracted the antidepressant-like actions of ketamine and (2R,6R)-HNK in the forced swim test. To conclude, by studying neuronally released glutamate, we identified a novel retrograde adenosinergic feedback mechanism that mediate inhibitory actions of ketamine on glutamate release that may contribute to its rapid antidepressant action.

Fluoxetine Suppresses Glutamate- and GABA-Mediated Neurotransmission by Altering SNARE Complex.

Major depressive disorder is one of the most common neuropsychiatric disorders worldwide. The treatment of choice that shows good efficacy in mood stabilization is based on selective serotonin reuptake inhibitors (SSRIs). Their primary mechanism of action is considered to be the increased synaptic concentration of serotonin through blockade of the serotonin transporter (SERT). In this study, we described an alternative mode of action of fluoxetine (FLX), which is a representative member of the SSRI class of antidepressants. We observed that FLX robustly decreases both glutamatergic and gamma-Aminobutyric acid (GABA)-ergic synaptic release in a SERT-independent manner. Moreover, we showed that this effect may stem from the ability of FLX to change the levels of main components of the SNARE (solubile N-ethylmaleimide-sensitive factor attachment protein receptor) complex. Our data suggest that this downregulation of SNARE fusion machinery involves diminished activity of protein kinase C (PKC) due to FLX-induced blockade of P/Q type of voltage-gated calcium channels (VGCCs). Taken together, by virtue of its inhibition at SERT, fluoxetine increases extracellular serotonin levels; however, at the same time, by reducing SNARE complex function, this antidepressant reduces glutamate and GABA release.

Riluzole attenuates the efficacy of glutamatergic transmission by interfering with the size of the readily releasable neurotransmitter pool.

Riluzole is a potent neuroprotective agent which primarily inhibits excitatory neurotransmission interfering with presynaptic release, uptake and postsynaptic actions of glutamate by mechanisms that are not well understood. Riluzole and related prodrugs with improved blood brain barrier penetrance, are shown to be effective for the treatment of amyotrophic lateral sclerosis, ataxias, epilepsy and mood disorders. Our study was undertaken to decipher molecular and subcellular mechanisms of riluzole's antiglutamatergic effect, particularly focusing on presynaptic active zone structure and function. Applying multifarious live cell imaging techniques and amperometric glutamate recordings, we measured the impact of riluzole on presynaptic activity, synaptic vesicle recycling and glutamate release. Our in vitro and in vivo data revealed a unique mechanism whereby riluzole reduces the efficacy of glutamatergic transmission by selectively lowering the size of the readily releasable pool. This effect was correlated with the inhibition of protein kinase C-dependent Munc18-1 phosphorylation which is known to interfere with neurotransmitter release.

Physiological Concentrations of Amyloid Beta Regulate Recycling of Synaptic Vesicles via Alpha7 Acetylcholine Receptor and CDK5/Calcineurin Signaling.

Despite the central role of amyloid ß (Aß) peptide in the etiopathogenesis of Alzheimer's disease (AD), its physiological function in healthy brain is still debated. It is well established that elevated levels of Aß induce synaptic depression and dismantling, connected with neurotoxicity and neuronal loss. Growing evidence suggests a positive regulatory effect of Aß on synaptic function and cognition; however the exact cellular and molecular correlates are still unclear. In this work, we tested the effect of physiological concentrations of Aß species of endogenous origin on neurotransmitter release in rat cortical and hippocampal neurons grown in dissociated cultures. Modulation of production and degradation of the endogenous Aß species as well as applications of the synthetic rodent Aß40 and Aß42 affected efficacy of neurotransmitter release from individual presynapses. Low picomolar Aß40 and Aß42 increased, while Aß depletion or application of low micromolar concentration decreased synaptic vesicle recycling, showing a hormetic effect of Aß on neurotransmitter release. These Aß-mediated modulations required functional alpha7 acetylcholine receptors as well as extracellular and intracellular calcium, involved regulation of CDK5 and calcineurin signaling and increased recycling of synaptic vesicles. These data indicate that Aß regulates neurotransmitter release from presynapse and suggest that failure of the normal physiological function of Aß in the fine-tuning of SV cycling could disrupt synaptic function and homeostasis, which would, eventually, lead to cognitive decline and neurodegeneration.

Molecular mechanisms driving homeostatic plasticity of neurotransmitter release.

Homeostatic plasticity is a process by which neurons adapt to the overall network activity to keep their firing rates in a reasonable range. At the cellular level this kind of plasticity comprises modulation of cellular excitability and tuning of synaptic strength. In this review we concentrate on presynaptic homeostatic plasticity controlling the efficacy of neurotransmitter release from presynaptic boutons. While morphological and electrophysiological approaches were successful to describe homeostatic plasticity-induced changes in the presynaptic architecture and function, cellular and molecular mechanisms underlying those modifications remained largely unknown for a long time. We summarize the latest progress made in the understanding of homeostasis-induced regulation of different steps of the synaptic vesicle cycle and the molecular machineries involved in this process. We particularly focus on the role of presynaptic scaffolding proteins, which functionally and spatially organize synaptic vesicle clusters, neurotransmitter release sites and the associated endocytic machinery. These proteins turned out to be major presynaptic substrates for remodeling during homeostatic plasticity. Finally, we discuss cellular processes and signaling pathways acting during homeostatic molecular remodeling and their potential involvement in the maladaptive plasticity occurring in multiple neuropathologic conditions such as neurodegeneration, epilepsy and neuropsychiatric disorders.

[Financial sustainability of home care in the health system of the Republic of Serbia].

INTRODUCTION: Over the last several years, during the economic crisis, the Ministry of Health and the Republican Health Insurance Fund (RHIF) have been faced with new challenges in the sphere of healthcare services financing both in the primary as well as other types of health insurance in the Republic of Serbia (RS). OBJECTIVE: Analysis of cost-effectiveness of two models of organization of home treatment and healthcare in the primary insurance, with evaluation of the cost sustainability of a single visit by the in-home therapy team. METHODS: Economic evaluation of the cost of home treatment and healthcare provision in 2011 was performed. In statistical analysis, the methods of descriptive statistics were employed. The structure of fixed costs of home healthcare was developed according to the RS official norms, as well as fixed costs of providing services of home therapy by the Healthcare Centre "New Belgrade". The statement of account for provided home therapy services was made utilizing the RHIF price list. RESULTS: The results showed that the cost of home healthcare and therapy of the heterogeneous population of patients in the Healthcare Centre "New Belgrade" was more cost-effective in relation to the cost of providing home therapy services according to the RS official norms. CONCLUSION: Approved costs utilized when making a contract for services of home therapy and healthcare with the RHIF are not financially sustainable. It was shown that the price of 10 EUR for each home visit by the in-home therapy team enables sustainability of this form of providing healthcare services in RS.

Differential spatial expression and subcellular localization of CtBP family members in rodent brain.

C-terminal binding proteins (CtBPs) are well-characterized nuclear transcriptional co-regulators. In addition, cytoplasmic functions were discovered for these ubiquitously expressed proteins. These include the involvement of the isoform CtBP1-S/BARS50 in cellular membrane-trafficking processes and a role of the isoform RIBEYE as molecular scaffolds in ribbons, the presynaptic specializations of sensory synapses. CtBPs were suggested to regulate neuronal differentiation and they were implied in the control of gene expression during epileptogenesis. However, the expression patterns of CtBP family members in specific brain areas and their subcellular localizations in neurons in situ are largely unknown. Here, we performed comprehensive assessment of the expression of CtBP1 and CtBP2 in mouse brain at the microscopic and the ultra-structural levels using specific antibodies. We quantified and compared expression levels of both CtBPs in biochemically isolated brain fractions containing cellular nuclei or synaptic compartment. Our study demonstrates differential regional and subcellular expression patterns for the two CtBP family members in brain and reveals a previously unknown synaptic localization for CtBP2 in particular brain regions. Finally, we propose a mechanism of differential synapto-nuclear targeting of its splice variants CtBP2-S and CtBP2-L in neurons.

Extensive remodeling of the presynaptic cytomatrix upon homeostatic adaptation to network activity silencing.

Global changes of activity in neuronal networks induce homeostatic adaptations of synaptic strengths, which involve functional remodeling of both presynaptic and postsynaptic apparatuses. Despite considerable advances in understanding cellular properties of homeostatic synaptic plasticity, the underlying molecular mechanisms are not fully understood. Here, we explored the hypothesis that adaptive homeostatic adjustment of presynaptic efficacy involves molecular remodeling of the release apparatus including the presynaptic cytomatrix, which spatially and functionally coordinates neurotransmitter release. We found significant downregulation of cellular expression levels of presynaptic scaffolding proteins Bassoon, Piccolo, ELKS/CAST, Munc13, RIM, liprin-α, and synapsin upon prolonged (48 h) activity depletion in rat neuronal cultures. This was accompanied by a general reduction of Bassoon, Piccolo, ELKS/CAST, Munc13, and synapsin levels at synaptic sites. Interestingly, RIM was upregulated in a subpopulation of synapses. At the level of individual synapses, RIM quantities correlated well with synaptic activity, and a constant relationship between RIM levels and synaptic activity was preserved upon silencing. Silencing also induced synaptic enrichment of other previously identified regulators of presynaptic release probability, i.e., synaptotagmin1, SV2B, and P/Q-type calcium channels. Seeking responsible cellular mechanisms, we revealed a complex role of the ubiquitin-proteasome system in the functional presynaptic remodeling and enhanced degradation rates of Bassoon and liprin-α upon silencing. Together, our data indicate a significant molecular reorganization of the presynaptic release apparatus during homeostatic adaptation to network inactivity and identify RIM, synaptotagmin1, Ca(v)2.1, and SV2B as molecular candidates underlying the main silencing-induced functional hallmark at presynapse, i.e., increase of neurotransmitter release probability.

Dynein light chain regulates axonal trafficking and synaptic levels of Bassoon.

Bassoon and the related protein Piccolo are core components of the presynaptic cytomatrix at the active zone of neurotransmitter release. They are transported on Golgi-derived membranous organelles, called Piccolo-Bassoon transport vesicles (PTVs), from the neuronal soma to distal axonal locations, where they participate in assembling new synapses. Despite their net anterograde transport, PTVs move in both directions within the axon. How PTVs are linked to retrograde motors and the functional significance of their bidirectional transport are unclear. In this study, we report the direct interaction of Bassoon with dynein light chains (DLCs) DLC1 and DLC2, which potentially link PTVs to dynein and myosin V motor complexes. We demonstrate that Bassoon functions as a cargo adapter for retrograde transport and that disruption of the Bassoon-DLC interactions leads to impaired trafficking of Bassoon in neurons and affects the distribution of Bassoon and Piccolo among synapses. These findings reveal a novel function for Bassoon in trafficking and synaptic delivery of active zone material.

Continuous infusion of ceftazidime in critically ill patients undergoing continuous venovenous haemodiafiltration: pharmacokinetic evaluation and dose recommendation.

INTRODUCTION: In seriously infected patients with acute renal failure and who require continuous renal replacement therapy, data on continuous infusion of ceftazidime are lacking. Here we analyzed the pharmacokinetics of ceftazidime administered by continuous infusion in critically ill patients during continuous venovenous haemodiafiltration (CVVHDF) in order to identify the optimal dosage in this setting. METHOD: Seven critically ill patients were prospectively enrolled in the study. CVVHDF was performed using a 0.6 m2 AN69 high-flux membrane and with blood, dialysate and ultrafiltration flow rates of 150 ml/min, 1 l/hour and 1.5 l/hour, respectively. Based on a predicted haemodiafiltration clearance of 32.5 ml/min, all patients received a 2 g loading dose of ceftazidime, followed by a 3 g/day continuous infusion for 72 hours. Serum samples were collected at 0, 3, 15 and 30 minutes and at 1, 2, 4, 6, 8, 12, 24, 36, 48 and 72 hours; dialysate/ultrafiltrate samples were taken at 2, 8, 12, 24, 36 and 48 hours. Ceftazidime concentrations in serum and dialysate/ultrafiltrate were measured using high-performance liquid chromatography. RESULTS: The mean (+/- standard deviation) elimination half-life, volume of distribution, area under the concentration-time curve from time 0 to 72 hours, and total clearance of ceftazidime were 4 +/- 1 hours, 19 +/- 6 l, 2514 +/- 212 mg/h per l, and 62 +/- 5 ml/min, respectively. The mean serum ceftazidime steady-state concentration was 33.5 mg/l (range 28.8-36.3 mg/l). CVVHDF effectively removed continuously infused ceftazidime, with a sieving coefficient and haemodiafiltration clearance of 0.81 +/- 0.11 and 33.6 +/- 4 mg/l, respectively. CONCLUSION: We conclude that a dosing regimen of 3 g/day ceftazidime, by continuous infusion, following a 2 g loading dose, results in serum concentrations more than four times the minimum inhibitory concentration for all susceptible pathogens, and we recommend this regimen in critically ill patients undergoing CVVHDF.