Reactivation of chick erythrocyte nuclei after fusion with enucleated cells.

Inactivated Sendai virus was used to fuse nucleated chick erythrocytes with mouse L and A9 cells which had been enucleated by centrifugation in the presence of cytochalasinB. The enucleation step removed the nuclei from more than 99% of the cells. During the fusion step, chick erythrocyte nuclei were introduced into 20% of the enucleated mouse cytoplasms. This resulted in the formation of a large number of "reconstituted cells" where practically all the cytoplasm originated from the mouse cell while the nucleus was of chick origin. The chick erythrocyte nuclei appeared to become well integrated into the mouse cytoplasms since they increased dramatically in size and dry mass, formed nucleolus-like bodies, and resumed RNA synthesis. This, however, did not prevent a gradual decrease in the rate of protein synthesis in the cytoplasm after the removal of the mouse nucleus. Protein synthesis decayed at a similar rate in both reconstituted and enucleated cells. The majority of these "cells" died within 48 h and all of them within 5 days after enucleation/fusion. By contrast, the small number of L cells which failed to become enucleated multiplied rapidly. The results obtained suggest that the reactivation of the chick erythrocyte nuclei is not fast enough to rescue the enucleated mouse cytoplasms.     

Estimating the dimensionality of the manifold underlying multi-electrode neural recordings

It is generally accepted that the number of neurons in a given brain area far exceeds the number of neurons needed to carry any specific function controlled by that area. For example, motor areas of the human brain contain tens of millions of neurons that control the activation of tens or at most hundreds of muscles. This massive redundancy implies the covariation of many neurons, which constrains the population activity to a low-dimensional manifold within the space of all possible patterns of neural activity. To gain a conceptual understanding of the complexity of the neural activity within a manifold, it is useful to estimate its dimensionality, which quantifies the number of degrees of freedom required to describe the observed population activity without significant information loss. While there are many algorithms for dimensionality estimation, we do not know which are well suited for analyzing neural activity. The objective of this study was to evaluate the efficacy of several representative algorithms for estimating the dimensionality of linearly and nonlinearly embedded data. We generated synthetic neural recordings with known intrinsic dimensionality and used them to test the algorithms’ accuracy and robustness. We emulated some of the important challenges associated with experimental data by adding noise, altering the nature of the embedding of the low-dimensional manifold within the high-dimensional recordings, varying the dimensionality of the manifold, and limiting the amount of available data. We demonstrated that linear algorithms overestimate the dimensionality of nonlinear, noise-free data. In cases of high noise, most algorithms overestimated the dimensionality. We thus developed a denoising algorithm based on deep learning, the “Joint Autoencoder”, which significantly improved subsequent dimensionality estimation. Critically, we found that all algorithms failed when the intrinsic dimensionality was high (above 20) or when the amount of data used for estimation was low. Based on the challenges we observed, we formulated a pipeline for estimating the dimensionality of experimental neural data.     

Insertion of Alzheimer's A beta 40 peptide into lipid monolayers

The amyloid beta (A beta) peptide is the major component found in the amyloid deposits in the brains of Alzheimer's disease patients. In vitro studies have demonstrated that the aggregation of A beta can take place at three orders of magnitude lower concentrations in the presence of phospholipid molecules compared to bulk peptide studies, suggesting that membrane lipids may mediate A beta toxicity. To understand the interaction of A beta with lipid membranes, we have examined A beta 40 with anionic dipalmitoylphosphatidylglycerol (DPPG), zwitterionic dipalmitoylphosphatidylcholine (DPPC), and cationic dipalmitoyltrimethylammonium propane (DPTAP) monolayers under different subphase conditions. We have used a constant surface pressure insertion assay to assess the degree of peptide insertion into the lipids. Simultaneously, we monitored the surface morphology of the monolayers with fluorescence microscopy. We have also performed dual-probe fluorescence measurements where both the peptide and lipid are tagged with chromophores. Isotherm measurements show that A beta inserts into both DPTAP and DPPG monolayers under physiologically relevant conditions. Insertion into DPPC occurs at lipid densities below that found in a bilayer. The level of insertion is inversely proportional to the lipid packing density. Our results indicate that lipids need not be anionic to interact with A beta. Electrostatic effects involved in A beta 40-lipid interaction are discussed.     

Viability of cells reconstituted by virus-induced fusion of minicells with anucleate cells

The addition of 1 mM ATP to rabbit peritoneal polymorphonuclear leukocytes suspended in a solution of glycerol causes a decrease in their volume by 4–17% within 3 min. The suspending medium must not only contain glycerol but be of low ionic strength for ATP to be effective. Divalent cations are also required. Ca²⁺ present alone can sustain the volume increase induced by ATP; in the presence of low concentrations of Ca²⁺, Mg²⁺ is also effective but not to the same extent as Ca²⁺. When the cell volume is contracted by the ATP in the presence of Ca²⁺ the addition of EGTA induces a reexpansion of the volume.The organic mercurial, salyrgan prevents the ATP induced reduction in the volume but ouabain has no effect. Guanosine triphosphate (GTP), uridine triphosphate (UTP), and adenosine diphosphate (ADP) can also decrease the volume of the glycerinated leukocytes but to a distinctly lesser extent than ATP. Adenosine monophosphate (AMP) and cyclic adenosine monophosphate (cAMP) are without significant effect.The results indicate that the volume decrease caused by exogenous ATP is unlikely to be a passive osmotic or an active, ouabain-sensitive process. The similarities to the interaction of ATP with actomyosin suggest that the volume decrease might be a result of the contraction of the leukocyte actomyosin by ATP.     

Molecular Underpinnings of Synaptic Vesicle Pool Heterogeneity

Neuronal communication relies on chemical synaptic transmission for information transfer and processing. Chemical neurotransmission is initiated by synaptic vesicle fusion with the presynaptic active zone resulting in release of neurotransmitters. Classical models have assumed that all synaptic vesicles within a synapse have the same potential to fuse under different functional contexts. In this model, functional differences among synaptic vesicle populations are ascribed to their spatial distribution in the synapse with respect to the active zone. Emerging evidence suggests, however, that synaptic vesicles are not a homogenous population of organelles, and they possess intrinsic molecular differences and differential interaction partners. Recent studies have reported a diverse array of synaptic molecules that selectively regulate synaptic vesicles' ability to fuse synchronously and asynchronously in response to action potentials or spontaneously irrespective of action potentials. Here we discuss these molecular mediators of vesicle pool heterogeneity that are found on the synaptic vesicle membrane, on the presynaptic plasma membrane, or within the cytosol and consider some of the functional consequences of this diversity. This emerging molecular framework presents novel avenues to probe synaptic function and uncover how synaptic vesicle pools impact neuronal signaling.      

Vti1a identifies a vesicle pool that preferentially recycles at rest and maintains spontaneous neurotransmission

Recent studies suggest that synaptic vesicles (SVs) giving rise to spontaneous neurotransmission are distinct from those that carry out evoked release. However, the molecular basis of this dichotomy remains unclear. Here, we focused on two noncanonical SNARE molecules, Vps10p-tail-interactor-1a (vti1a) and VAMP7, previously shown to reside on SVs. Using simultaneous multicolor imaging at individual synapses, we could show that compared to the more abundant vesicular SNARE synaptobrevin2, both vti1a and VAMP7 were reluctantly mobilized during activity. Vti1a, but not VAMP7, showed robust trafficking under resting conditions that could be partly matched by synaptobrevin2. Furthermore, loss of vti1a function selectively reduced high-frequency spontaneous neurotransmitter release detected postsynaptically. Expression of a truncated version of vti1a augmented spontaneous release more than full-length vti1a, suggesting that an autoinhibitory process regulates vti1a function. Taken together, these results support the premise that in its native form vti1a selectively maintains spontaneous neurotransmitter release.     

Retrospective analyses of serum lipids and lipoproteins and severity of disease in 60Co-irradiated Sus scrofa domestica and Macaca mulatta

Profound neutropenia that provides an opportunity for infections to develop into sepsis remains an important cause of morbidity and mortality in patients after irradiation. Human clinical studies find extremely low concentrations of cholesterol (less than 120 mg/dl) associated with high risk of death in critically ill adult patients admitted to intensive care units. This retrospective study was initiated as part of separate investigations of radiation-induced acquired infections in 2 large animal species receiving high-dose whole-body irradiation from a 60Co gamma-photon source. Nine Yucatan minipigs (Sus scrofa domestica) and 16 rhesus macaques (Macaca mulatta) were evaluated for sepsis, serum lipid and lipoprotein concentrations, and other blood parameters. For each species, animals were grouped into 2 categories--septic and nonseptic--and severity of disease was quantified by use of a scoring system. Significantly lower high-density lipoprotein (HDL) concentrations were found in the septic pigs at 24 and 48 h as compared with nonseptic pigs. HDL was significantly decreased in septic macaques within 24 h and 3 to 4, 6 to 7, and 9 to 10 d after diagnosis of sepsis, compared with that in nonseptic macaques. Coupled with hypocholesterolemia, decreased serum HDL was the parameter that was associated with disease severity at the time of sampling. Our data indicate that HDL is a reliable marker for severity of disease in these 2 preclinical models of irradiation-induced sepsis.     

Presynaptic unsilencing: searching for a mechanism

Nascent synaptic networks have a high incidence of silent synapses. In this issue of Neuron, Shen et al. show that a brief burst of action potentials rapidly awaken silent synapses by increasing the availability of synaptic vesicles for fusion through BDNF-triggered presynaptic actin remodeling mediated by the small GTPase Cdc42.     

Synaptotagmin 7 splice variants differentially regulate synaptic vesicle recycling

The speed of synaptic vesicle recycling determines the efficacy of neurotransmission during repetitive stimulation. Synaptotagmins are synaptic C(2)-domain proteins that are involved in exocytosis, but have also been linked to endocytosis. We now demonstrate that upon expression in transfected neurons, a short splice variant of synaptotagmin 7 that lacks C(2)-domains accelerates endocytic recycling of synaptic vesicles, whereas a longer splice variant that contains C(2)-domains decelerates recycling. These results suggest that alternative splicing of synaptotagmin 7 acts as a molecular switch, which targets vesicles to fast and slow recycling pathways.