Comparison of Cardiovascular Medicines Prices in Four European Countries.

The aim of the current study was to compare pricing methodologies at the manufacturer, wholesale, and retail levels, and to estimate the price differences of AT1-receptor blockers (sartans), Angiotensin-converting enzyme (ACE)-inhibitors, and their fixed-dose combinations (FDCs) in four countries using similar methodologies: Slovakia, Greece, Bulgaria, and Romania (SK, GR, BG, and RO, respectively). The methodologies for manufacturer, wholesale, and retail price establishment have been compared using nationally implemented rules. Overlapping trademarks were established retrospectively on the manufacturer and retail levels in November 2017. The average price per tablet, percentage of price deviation, and statistically significant differences were calculated. The selected countries apply external reference pricing at the manufacturer level. A wide variation in the number of referent countries was observed (from 12 to 27). Despite the use of a regressive scale for price calculation, large variations between margins and value-added tax (VAT) are established, thus leading to different final medicine prices. This study found that medicine prices were lower in RO than in other selected countries. It was caused by the fact that 15 products had the lowest manufacturer price and 14 products had the lowest retail price in RO. Results of Kruskal-Wallis test showed that there were no significant differences between prices per tablet on the manufacturer and retail levels. In the group of fixed-dose combinations, ramipril/hydrochlorothiazide, and irbesartan/hydrochlorothiazide showed more than 100% deviation. The prices of cardiovascular medicines differed within the observed countries. The differences in pricing methodologies (e.g., margins, VAT) at the national level did not significantly affect retail prices, as a low manufacturer price usually leads to a low retail price.

Photooxidation-guided ultrastructural identification and analysis of cells in neuronal tissue labeled with green fluorescent protein.

The ultrastructural characterization of neuronal compartments in intact tissue labeled with green fluorescent protein (GFP) remains a frequently encountered challenge, despite work establishing photooxidation of GFP in cultured cells. However, most applications require the detection of GFP or GFP fusion proteins expressed in intact tissue. Here, we report that illumination of GFP variants in oxygen-enriched environment reliably generated electron-dense 3,3'-diaminobenzidine (DAB) precipitates in slices from rat brain. The method is applicable to GFP variants tagged to presynaptic proteins as well as to soluble GFP in various brain regions. Serial section scanning electron microscopy was used to examine genetically labeled presynaptic terminals at high resolution and to generate three-dimensional representations of the synapses. Thus, we introduce a generally applicable correlative approach for the identification of presynaptic terminals genetically labeled with green fluorescent proteins in tissue slices and their ultrastructural characterization.

Overexpression of synapsin Ia in the rat calyx of Held accelerates short-term plasticity and decreases synaptic vesicle volume and active zone area.

Synapsins are synaptic vesicle (SV) proteins organizing a component of the reserve pool of vesicles at most central nervous system synapses. Alternative splicing of the three mammalian genes results in multiple isoforms that may differentially contribute to the organization and maintenance of the SV pools. To address this, we first characterized the expression pattern of synapsin isoforms in the rat calyx of Held. At postnatal day 16, synapsins Ia, Ib, IIb and IIIa were present, while IIa-known to sustain repetitive transmission in glutamatergic terminals-was not detectable. To test if the synapsin I isoforms could mediate IIa-like effect, and if this depends on the presence of the E-domain, we overexpressed either synapsin Ia or synapsin Ib in the rat calyx of Held via recombinant adeno-associated virus-mediated gene transfer. Although the size and overall structure of the perturbed calyces remained unchanged, short-term depression and recovery from depression were accelerated upon overexpression of synapsin I isoforms. Using electron microscopic three-dimensional reconstructions we found a redistribution of SV clusters proximal to the active zones (AZ) alongside with a decrease of both AZ area and SV volume. The number of SVs at individual AZs was strongly reduced. Hence, our data indicate that the amount of synapsin Ia expressed in the calyx regulates the rate and extent of short-term synaptic plasticity by affecting vesicle recruitment to the AZ. Finally, our study reveals a novel contribution of synapsin Ia to define the surface area of AZs.

Synapsin-dependent reserve pool of synaptic vesicles supports replenishment of the readily releasable pool under intense synaptic transmission.

Synapsins are abundant synaptic vesicle (SV)-associated proteins thought to mediate synaptic vesicle mobility and clustering at most synapses. We used synapsin triple knock-out (TKO) mice to examine the morphological and functional consequences of deleting all synapsin isoforms at the calyx of Held, a giant glutamatergic synapse located in the auditory brain stem. Quantitative three-dimensional (3D) immunohistochemistry of entire calyces showed lower amounts of the synaptic vesicle protein vGluT1 while the level of the active zone marker bassoon was unchanged in TKO terminals. Examination of brain lysates by ELISA revealed a strong reduction in abundance of several synaptic vesicle proteins, while proteins of the active zone cytomatrix or postsynaptic density were unaffected. Serial section scanning electron microscopy of large 3D-reconstructed segments confirmed a decrease in the number of SVs to approximately 50% in TKO calyces. Short-term depression tested at stimulus frequencies ranging from 10 to 300 Hz was accelerated only at frequencies above 100 Hz and the time course of recovery from depression was slowed in calyces lacking synapsins. These results reveal that in wild-type synapses, the synapsin-dependent reserve pool contributes to the replenishment of the readily releasable pool (RRP), although accounting only for a small fraction of the SVs that enter the RRP. In conclusion, our results suggest that synapsins may be required for normal synaptic vesicle biogenesis, trafficking and immobilization of synaptic vesicles, yet they are not essential for sustained high-frequency synaptic transmission at the calyx terminal.

Inhibition of exocytosis or endocytosis blocks activity-dependent redistribution of synapsin.

The synaptic vesicle cycle encompasses the pre-synaptic events that drive neurotransmission. Influx of calcium leads to the fusion of synaptic vesicles with the plasma membrane and the release of neurotransmitter, closely followed by endocytosis. Vacated release sites are repopulated with vesicles which are then primed for release. When activity is intense, reserve vesicles may be mobilized to counteract an eventual decline in transmission. Recently, interplay between endocytosis and repopulation of the readily releasable pool of vesicles has been identified. In this study, we show that exo-endocytosis is necessary to enable detachment of synapsin from reserve pool vesicles during synaptic activity. We report that blockage of exocytosis in cultured mouse hippocampal neurons, either by tetanus toxin or by the deletion of munc13, inhibits the activity-dependent redistribution of synapsin from the pre-synaptic terminal into the axon. Likewise, perturbation of endocytosis with dynasore or by a dynamin dominant-negative mutant fully prevents synapsin redistribution. Such inhibition of synapsin redistribution occurred despite the efficient phosphorylation of synapsin at its protein kinase A/CaMKI site, indicating that disengagement of synapsin from the vesicles requires exocytosis and endocytosis in addition to phosphorylation. Our results therefore reveal hitherto unidentified feedback within the synaptic vesicle cycle involving the synapsin-managed reserve pool.