Ultrastructural and functional fate of recycled vesicles in hippocampal synapses

Efficient recycling of synaptic vesicles is thought to be critical for sustained information transfer at central terminals. However, the specific contribution that retrieved vesicles make to future transmission events remains unclear. Here we exploit fluorescence and time-stamped electron microscopy to track the functional and positional fate of vesicles endocytosed after readily releasable pool (RRP) stimulation in rat hippocampal synapses. We show that most vesicles are recovered near the active zone but subsequently take up random positions in the cluster, without preferential bias for future use. These vesicles non-selectively queue, advancing towards the release site with further stimulation in an actin-dependent manner. Nonetheless, the small subset of vesicles retrieved recently in the stimulus train persist nearer the active zone and exhibit more privileged use in the next RRP. Our findings reveal heterogeneity in vesicle fate based on nanoscale position and timing rules, providing new insights into the origins of future pool constitution

Vaccines against toxoplasma gondii : challenges and opportunities

Development of vaccines against Toxoplasma gondii infection in humans is of high priority, given the high burden of disease in some areas of the world like South America, and the lack of effective drugs with few adverse effects. Rodent models have been used in research on vaccines against T. gondii over the past decades. However, regardless of the vaccine construct, the vaccines have not been able to induce protective immunity when the organism is challenged with T. gondii, either directly or via a vector. Only a few live, attenuated T. gondii strains used for immunization have been able to confer protective immunity, which is measured by a lack of tissue cysts after challenge. Furthermore, challenge with low virulence strains, especially strains with genotype II, will probably be insufficient to provide protection against the more virulent T. gondii strains, such as those with genotypes I or II, or those genotypes from South America not belonging to genotype I, II or III. Future studies should use animal models besides rodents, and challenges should be performed with at least one genotype II T. gondii and one of the more virulent genotypes. Endpoints like maternal-foetal transmission and prevention of eye disease are important in addition to the traditional endpoint of survival or reduction in numbers of brain cysts after challenge

Dynamics of Multiple Trafficking Behaviors of Individual Synaptic Vesicles Revealed by Quantum-Dot Based Presynaptic Probe

Although quantum dots (QDs) have provided invaluable information regarding the diffusive behaviors of postsynaptic receptors, their application in presynaptic terminals has been rather limited. In addition, the diffraction-limited nature of the presynaptic bouton has hampered detailed analyses of the behaviors of synaptic vesicles (SVs) at synapses. Here, we created a quantum-dot based presynaptic probe and characterized the dynamic behaviors of individual SVs. As previously reported, the SVs exhibited multiple exchanges between neighboring boutons. Actin disruption induced a dramatic decrease in the diffusive behaviors of SVs at synapses while microtubule disruption only reduced extrasynaptic mobility. Glycine-induced synaptic potentiation produced significant increases in synaptic and inter-boutonal trafficking of SVs, which were NMDA receptor- and actin-dependent while NMDA-induced synaptic depression decreased the mobility of the SVs at synapses. Together, our results show that sPH-AP-QD revealed previously unobserved trafficking properties of SVs around synapses, and the dynamic modulation of SV mobility could regulate presynaptic efficacy during synaptic activity

Computational geometry analysis of dendritic spines by structured illumination microscopy

We are currently short of methods that can extract objective parameters of dendritic spines useful for their categorization. Authors present in this study an automatic analytical pipeline for spine geometry using 3D-structured illumination microscopy, which can effectively extract many geometrical parameters of dendritic spines without bias and automatically categorize spine population based on their morphological feature

Synapse Clusters Are Preferentially Formed by Synapses with Large Recycling Pool Sizes

Synapses are distributed heterogeneously in neural networks. The relationship between the spatial arrangement of synapses and an individual synapse's structural and functional features remains to be elucidated. Here, we examined the influence of the number of adjacent synapses on individual synaptic recycling pool sizes. When measuring the discharge of the styryl dye FM1–43 from electrically stimulated synapses in rat hippocampal tissue cultures, a strong positive correlation between the number of neighbouring synapses and recycling vesicle pool sizes was observed. Accordingly, vesicle-rich synapses were found to preferentially reside next to neighbours with large recycling pool sizes. Although these synapses with large recycling pool sizes were rare, they were densely arranged and thus exhibited a high amount of release per volume. To consolidate these findings, functional terminals were marked by live-cell antibody staining with anti-synaptotagmin-1-cypHer or overexpression of synaptopHluorin. Analysis of synapse distributions in these systems confirmed the results obtained with FM 1–43. Our findings support the idea that clustering of synapses with large recycling pool sizes is a distinct developmental feature of newly formed neural networks and may contribute to functional plasticity

Alternatives to vitamin B 1 uptake revealed with discovery of riboswitches in multiple marine eukaryotic lineages

Vitamin B 1 (thiamine pyrophosphate, TPP) is essential to all life but scarce in ocean surface waters. In many bacteria and a few eukaryotic groups thiamine biosynthesis genes are controlled by metabolite-sensing mRNA-based gene regulators known as riboswitches. Using available genome sequences and transcriptomes generated from ecologically important marine phytoplankton, we identified 31 new eukaryotic riboswitches. These were found in alveolate, cryptophyte, haptophyte and rhizarian phytoplankton as well as taxa from two lineages previously known to have riboswitches (green algae and stramenopiles). The predicted secondary structures bear hallmarks of TPP-sensing riboswitches. Surprisingly, most of the identified riboswitches are affiliated with genes of unknown function, rather than characterized thiamine biosynthesis genes. Using qPCR and growth experiments involving two prasinophyte algae, we show that expression of these genes increases significantly under vitamin B 1 -deplete conditions relative to controls. Pathway analyses show that several algae harboring the uncharacterized genes lack one or more enzymes in the known TPP biosynthesis pathway. We demonstrate that one such alga, the major primary producer Emiliania huxleyi, grows on 4-amino-5-hydroxymethyl-2-methylpyrimidine (a thiamine precursor moiety) alone, although long thought dependent on exogenous sources of thiamine. Thus, overall, we have identified riboswitches in major eukaryotic lineages not known to undergo this form of gene regulation. In these phytoplankton groups, riboswitches are often affiliated with widespread thiamine-responsive genes with as yet uncertain roles in TPP pathways. Further, taxa with 'incomplete' TPP biosynthesis pathways do not necessarily require exogenous vitamin B 1, making vitamin control of phytoplankton blooms more complex than the current paradigm suggests. © 2014 International Society for Microbial Ecology. All rights reserved

An ultrastructural readout of fluorescence recovery after photobleaching using correlative light and electron microscopy.

Fluorescence recovery after photobleaching (FRAP) provides an important quantitative readout of the mobility of fluorescently tagged structures in live tissue. Here we present a protocol for visualizing FRAP signal at the ultrastructural level, permitting the nature of recovered fluorescence signal to be studied at greater resolution than afforded by conventional light microscopy. Specifically we use FRAP, fixation, photoconversion and correlative light and electron microscopy (CLEM) to examine the ultrastructural organization of mobile FM1-43-labeled vesicles in synapses of cultured hippocampal neurons. At photobleached synapses, the FRAP signal can be visualized as photoconverted electron-dense vesicles. The combination of FRAP and CLEM provides a powerful tool for examining the specific localization of imported vesicles in relation to synaptic architecture. Moreover, with the increasing availability of photoconvertible fluorophores, this approach should be readily applicable to other systems where an ultrastructural characterization of FRAP signal is desirable. After cultures are prepared and ready to use, this protocol takes 2¿3 days