Inhibitory Synaptic Plasticity - Spike timing dependence and putative network function.

While the plasticity of excitatory synaptic connections in the brain has been widely studied, the plasticity of inhibitory connections is much less understood. Here, we present recent experimental and theoretical □ndings concerning the rules of spike timing-dependent inhibitory plasticity and their putative network function. This is a summary of a workshop at the COSYNE conference 2012.

Double synaptic weight neuron theory and its application

In this paper, a novel mathematical model of neuron-Double Synaptic Weight Neuron (DSWN)(l) is presented. The DSWN can simulate many kinds of neuron architectures, including Radial-Basis-Function (RBF), Hyper Sausage and Hyper Ellipsoid models, etc. Moreover, this new model has been implemented in the new CASSANN-II neurocomputer that can be used to form various types of neural networks with multiple mathematical models of neurons. The flexibility of the DSWN has also been described in constructing neural networks. Based on the theory of Biomimetic Pattern Recognition (BPR) and high-dimensional space covering, a recognition system of omni directionally oriented rigid objects on the horizontal surface and a face recognition system had been implemented on CASSANN-II neurocomputer. In these two special cases, the result showed DSWN neural network had great potential in pattern recognition.

Discussion on the basic mathematical models of neurons - Double synaptic weight neuron in general-purpose neurocomputer, theory and application

Based on the introduction of the traditional mathematical models of neurons in general-purpose neurocomputer, a novel all-purpose mathematical model-Double synaptic weight neuron (DSWN) is presented, which can simulate all kinds of neuron architectures, including Radial-Basis-Function (RBF) and Back-propagation (BP) models, etc. At the same time, this new model is realized using hardware and implemented in the new CASSANN-II neurocomputer that can be used to form various types of neural networks with multiple mathematical models of neurons. In this paper, the flexibility of the new model has also been described in constructing neural networks and based on the theory of Biomimetic pattern recognition (BPR) and high-dimensional space covering, a recognition system of omni directionally oriented rigid objects on the horizontal surface and a face recognition system had been implemented on CASSANN-H neurocomputer. The result showed DSWN neural network has great potential in pattern recognition.

Folate-Conjugated Micelles and Their Folate-Receptor-Mediated Endocytosis

A folate-conjugated copolymer PEG-PLA-PLL/folate was synthesized and mixed with pure PEG-PLA-PLL and a fluorescent model drug mFITC to prepare folate-conjugated micelles. The distribution of micelles was studied on cancer-cell-bearing mice via frozen slicing. The results show that mFITC is successfully encapsulated into folate(+) and folate(-)micelles; PEG-PLA-PLL micelles the latter can be internalized by both HeLa and CHO cells without selectivity due to their cationic surface charges, while folate(+)micelles exhibit more preferential endocytosis by HeLa cells than by CHO cells. The folate(-)micelles showed retention in both organs and tumors. The folate(+)micelles are a promising active targeting drug delivery system for FR over-expressing cells and they accumulate in tumor beds.

Conformational transition of DNA induced by cationic lipid vesicle in acidic solution: spectroscopy investigation

The conformational transition of DNA induced by the interaction between DNA and a cationic lipid vesicle, didodecyidimethylammonium bromide (DDAB), had been investigated by circular dichroism (CD) and UV spectroscopy methods. We used singular value decomposition least squares method (SVDLS) to analyze the experimental CD spectra. Although pH value influenced the conformation of DNA in solution, the results showed that upon binding to double helical DNA, positively charged liposomes induced a conformational transition of DNA molecules from the native B-form to more compact conformations. At the same time, no obvious conformational changes occurred at single-strand DNA (ssDNA). While the cationic lipid vesicles and double-strand DNA (dsDNA) were mixed at a high molar ratio of DDAB vesicles to dsDNA, the conformation of dsDNA transformed from the B-form to the C-form resulting in an increase in duplex stability (DeltaT(m) = 8 +/- 0.4 degreesC). An increasing in T-m was also observed while the cationic lipid vesicles interacted with ssDNA.

Spatial Pattern Learning, Catastophic Forgetting and Optimal Rules of Synaptic Transmission

It is a neural network truth universally acknowledged, that the signal transmitted to a target node must be equal to the product of the path signal times a weight. Analysis of catastrophic forgetting by distributed codes leads to the unexpected conclusion that this universal synaptic transmission rule may not be optimal in certain neural networks. The distributed outstar, a network designed to support stable codes with fast or slow learning, generalizes the outstar network for spatial pattern learning. In the outstar, signals from a source node cause weights to learn and recall arbitrary patterns across a target field of nodes. The distributed outstar replaces the outstar source node with a source field, of arbitrarily many nodes, where the activity pattern may be arbitrarily distributed or compressed. Learning proceeds according to a principle of atrophy due to disuse whereby a path weight decreases in joint proportion to the transmittcd path signal and the degree of disuse of the target node. During learning, the total signal to a target node converges toward that node's activity level. Weight changes at a node are apportioned according to the distributed pattern of converging signals three types of synaptic transmission, a product rule, a capacity rule, and a threshold rule, are examined for this system. The three rules are computationally equivalent when source field activity is maximally compressed, or winner-take-all when source field activity is distributed, catastrophic forgetting may occur. Only the threshold rule solves this problem. Analysis of spatial pattern learning by distributed codes thereby leads to the conjecture that the optimal unit of long-term memory in such a system is a subtractive threshold, rather than a multiplicative weight.

Outer Membrane Vesicle Production in Escherichia coli Relieves Envelope Stress and is Modulated by Changes in Peptidoglycan

Bacterial outer membrane vesicles (OMVs) are spherical buds of the outer membrane (OM) containing periplasmic lumenal components. OMVs have been demonstrated to play a critical part in the transmission of virulence factors, immunologically active compounds, and bacterial survival, however vesiculation also appears to be a ubiquitous physiological process for Gram-negative bacteria. Despite their characterized biological roles, especially for pathogens, very little is known about their importance for the originating organism as well as regulation and mechanism of production. Only when we have established their biogenesis can we fully uncover their roles in pathogenesis and bacterial physiology. The overall goal of this research was to characterize bacterial mutants which display altered vesiculation phenotypes using genetic and biochemical techniques, and thereby begin to elucidate the mechanism of vesicle production and regulation. One part of this work elucidated a synthetic genetic growth defect for a strain with reduced OMV production (ΔnlpA, inner membrane lipoprotein with a minor role in methionine transport) and envelope stress (ΔdegP, dual function periplasmic chaperone/ protease responsible for managing proteinaceous waste). This research showed that the growth defect of ΔnlpAΔdegP correlated with reduced OMV production with respect to the hyprevesiculator ΔdegP and the accumulation of protein in the periplasm and DegP substrates in the lumen of OMVs. We further demonstrated that OMVs do not solely act as a stress response pathway to rid the periplasm of otherwise damaging misfolded protein but also of accumulated peptidoglycan (PG) fragments and lipopolysaccharide (LPS), elucidating OMVs as a general stress response pathway critical for bacterial well-being. The second part of this work, focused on the role of PG structure, turnover and covalent crosslinks to the OM in vesiculation. We established a direct link between PG degradation and vesiculation: Mutations in the OM lipoprotein nlpI had been previously established as a very strong hypervesiculation phenotype. In the literature NlpI had been associated with another OM lipoprotein, Spr that was recently identified as a PG hydrolase. The data presented here suggest that NlpI acts as a negative regulator of Spr and that the ΔnlpI hypervesiculation phenotype is a result of rampantly degraded PG by Spr. Additionally, we found that changes in PG structure and turnover correlate with altered vesiculation levels, as well as non-canonical D-amino acids, which are secreted by numerous bacteria on the onset of stationary phase, being a natural factor to increase OMV production. Furthermore, we discovered an inverse relationship between the concentration of Lpp-mediated, covalent crosslinks and the level of OMV production under conditions of modulated PG metabolism and structure. In contrast, situations that lead to periplasmic accumulation (protein, PG fragments, and LPS) and consequent hypervesiculation the overall OM-PG crosslink concentration appears to be unchanged. Form this work, we conclude that multiple pathways lead to OMV production: Lpp concentration-dependent and bulk driven, Lpp concentration-independent.

NlpI-mediated modulation of outer membrane vesicle production through peptidoglycan dynamics in Escherichia coli.

Outer membrane vesicles (OMVs) are ubiquitously secreted from the outer membrane (OM) of Gram-negative bacteria. These heterogeneous structures are composed of OM filled with periplasmic content from the site of budding. By analyzing mutants that have vesicle production phenotypes, we can gain insight into the mechanism of OMV budding in wild-type cells, which has thus far remained elusive. In this study, we present data demonstrating that the hypervesiculation phenotype of the nlpI deletion mutant of Escherichia coli correlates with changes in peptidoglycan (PG) dynamics. Our data indicate that in stationary phase cultures the nlpI mutant exhibits increased PG synthesis that is dependent on spr, consistent with a model in which NlpI controls the activity of the PG endopeptidase Spr. In log phase, the nlpI mutation was suppressed by a dacB mutation, suggesting that NlpI regulates penicillin-binding protein 4 (PBP4) during exponential growth. The data support a model in which NlpI negatively regulates PBP4 activity during log phase, and Spr activity during stationary phase, and that in the absence of NlpI, the cell survives by increasing PG synthesis. Further, the nlpI mutant exhibited a significant decrease in covalent outer membrane (OM-PG) envelope stabilizing cross-links, consistent with its high level of OMV production. Based on these results, we propose that one mechanism wild-type Gram-negative bacteria can use to modulate vesiculation is by altering PG-OM cross-linking via localized modulation of PG degradation and synthesis.

Modulation of bacterial outer membrane vesicle production by envelope structure and content.

BACKGROUND: Vesiculation is a ubiquitous secretion process of Gram-negative bacteria, where outer membrane vesicles (OMVs) are small spherical particles on the order of 50 to 250 nm composed of outer membrane (OM) and lumenal periplasmic content. Vesicle functions have been elucidated in some detail, showing their importance in virulence factor secretion, bacterial survival, and biofilm formation in pathogenesis. Furthermore, OMVs serve as an envelope stress response, protecting the secreting bacteria from internal protein misfolding stress, as well as external envelope stressors. Despite their important functional roles very little is known about the regulation and mechanism of vesicle production. Based on the envelope architecture and prior characterization of the hypervesiculation phenotypes for mutants lacking the lipoprotein, Lpp, which is involved in the covalent OM-peptidoglycan (PG) crosslinks, it is expected that an inverse relationship exists between OMV production and PG-crosslinked Lpp. RESULTS: In this study, we found that subtle modifications of PG remodeling and crosslinking modulate OMV production, inversely correlating with bound Lpp levels. However, this inverse relationship was not found in strains in which OMV production is driven by an increase in "periplasmic pressure" resulting from the accumulation of protein, PG fragments, or lipopolysaccharide. In addition, the characterization of an nlpA deletion in backgrounds lacking either Lpp- or OmpA-mediated envelope crosslinks demonstrated a novel role for NlpA in envelope architecture. CONCLUSIONS: From this work, we conclude that OMV production can be driven by distinct Lpp concentration-dependent and Lpp concentration-independent pathways.

Stress-induced outer membrane vesicle production by Pseudomonas aeruginosa.

As an opportunistic Gram-negative pathogen, Pseudomonas aeruginosa must be able to adapt and survive changes and stressors in its environment during the course of infection. To aid survival in the hostile host environment, P. aeruginosa has evolved defense mechanisms, including the production of an exopolysaccharide capsule and the secretion of a myriad of degradative proteases and lipases. The production of outer membrane-derived vesicles (OMVs) serves as a secretion mechanism for virulence factors as well as a general bacterial response to envelope-acting stressors. This study investigated the effect of sublethal physiological stressors on OMV production by P. aeruginosa and whether the Pseudomonas quinolone signal (PQS) and the MucD periplasmic protease are critical mechanistic factors in this response. Exposure to some environmental stressors was determined to increase the level of OMV production as well as the activity of AlgU, the sigma factor that controls MucD expression. Overexpression of AlgU was shown to be sufficient to induce OMV production; however, stress-induced OMV production was not dependent on activation of AlgU, since stress caused increased vesiculation in strains lacking algU. We further determined that MucD levels were not an indicator of OMV production under acute stress, and PQS was not required for OMV production under stress or unstressed conditions. Finally, an investigation of the response of P. aeruginosa to oxidative stress revealed that peroxide-induced OMV production requires the presence of B-band but not A-band lipopolysaccharide. Together, these results demonstrate that distinct mechanisms exist for stress-induced OMV production in P. aeruginosa.

ARFGAP1 plays a central role in coupling COPI cargo sorting with vesicle formation.

Examining how key components of coat protein I (COPI) transport participate in cargo sorting, we find that, instead of ADP ribosylation factor 1 (ARF1), its GTPase-activating protein (GAP) plays a direct role in promoting the binding of cargo proteins by coatomer (the core COPI complex). Activated ARF1 binds selectively to SNARE cargo proteins, with this binding likely to represent at least a mechanism by which activated ARF1 is stabilized on Golgi membrane to propagate its effector functions. We also find that the GAP catalytic activity plays a critical role in the formation of COPI vesicles from Golgi membrane, in contrast to the prevailing view that this activity antagonizes vesicle formation. Together, these findings indicate that GAP plays a central role in coupling cargo sorting and vesicle formation, with implications for simplifying models to describe how these two processes are coupled during COPI transport.

ROLE OF ICAM-1-MEDIATED ENDOCYTOSIS IN ENDOTHELIAL FUNCTION AND IMPLICATIONS FOR CARRIER-ASSISTED DRUG DELIVERY

Intercellular adhesion molecule 1 (ICAM-1) is a transmembrane protein found on the surface of vascular endothelial cells (ECs). Its expression is upregulated at inflammatory sites, allowing for targeted delivery of therapeutics using ICAM-1-binding drug carriers. Engagement of multiple copies of ICAM-1 by these drug carriers induces cell adhesion molecule (CAM)-mediated endocytosis, which results in trafficking of carriers to lysosomes and across ECs. Knowledge about the regulation behind CAM-mediated endocytosis can help improve drug delivery, but questions remain about these regulatory mechanisms. Furthermore, little is known about the natural function of this endocytic pathway. To address these gaps in knowledge, we focused on two natural binding partners of ICAM-1 that potentially elicit CAM-mediated endocytosis: leukocytes (which bind ICAM-1 via β₂ integrins) and fibrin polymers (a main component of blood clots which binds ICAM-1 via the γ3 sequence). First, inspired by properties of these natural binding partners, we varied the size and targeting moiety of model drug carriers to determine how these parameters affect CAM-mediated endocytosis. Increasing ICAM-1-targeted carrier size slowed carrier uptake kinetics, reduced carrier trafficking to lysosomes, and increased carrier transport across ECs. Changing targeting moieties from antibodies to peptides decreased particle binding and uptake, lowered trafficking to lysosomes, and increased transport across ECs. Second, using cell culture models of leukocyte/EC interactions, inhibiting regulatory elements of the CAM-mediated pathway disrupted leukocyte sampling, a process crucial to leukocyte crossing of endothelial layers (transmigration). This inhibition also decreased leukocyte transmigration across ECs, specifically through the transcellular route, which occurs through a single EC without disassembly of cell-cell junctions. Third, fibrin meshes, which mimic blood clot fragments/remnants, bound to ECs at ICAM-1-enriched sites and were internalized by the endothelium. Inhibiting the CAM-mediated pathway disrupted this uptake. Following endocytosis, fibrin meshes trafficked to lysosomes where they were degraded. In mouse models, CAM-mediated endocytosis of fibrin meshes appeared to remove fibrin remnants at the endothelial surface, preventing re-initiation of the coagulation cascade. Overall, these results support a link between CAM-mediated endocytosis and leukocyte transmigration as well as uptake of fibrin materials by ECs. Furthermore, these results will guide the future design of ICAM-1-targeted carrier-assisted therapies.

The origin and evolution of synaptic proteins - choanoflagellates lead the way.

The origin of neurons was a key event in evolution, allowing metazoans to evolve rapid behavioral responses to environmental cues. Reconstructing the origin of synaptic proteins promises to reveal their ancestral functions and might shed light on the evolution of the first neuron-like cells in metazoans. By analyzing the genomes of diverse metazoans and their closest relatives, the evolutionary history of diverse presynaptic and postsynaptic proteins has been reconstructed. These analyses revealed that choanoflagellates, the closest relatives of metazoans, possess diverse synaptic protein homologs. Recent studies have now begun to investigate their ancestral functions. A primordial neurosecretory apparatus in choanoflagellates was identified and it was found that the mechanism, by which presynaptic proteins required for secretion of neurotransmitters interact, is conserved in choanoflagellates and metazoans. Moreover, studies on the postsynaptic protein homolog Homer revealed unexpected localization patterns in choanoflagellates and new binding partners, both which are conserved in metazoans. These findings demonstrate that the study of choanoflagellates can uncover ancient and previously undescribed functions of synaptic proteins.

LEARNING IMPULSE CONTROL IN A NOVEL ANIMAL MODEL: SYNAPTIC, CELLULAR, AND PHARMACOLOGICAL SUBSTRATES

Impulse control, an executive process that restrains inappropriate actions, is impaired in numerous psychiatric conditions. This thesis reports three experiments that utilized a novel animal model of impulse control, the response inhibition (RI) task, to examine the substrates that underlie learning this task. In the first experiment, rats were trained to withhold responding on the RI task, and then euthanized for electrophysiological testing. Training in the RI task increased the AMPA/NMDA ratio at the synapses of pyramidal neurons in the prelimbic, but not infralimbic, region of the medial prefrontal cortex. This enhancement paralleled performance as subjects underwent acquisition and extinction of the inhibitory response. AMPA/NMDA was elevated only in neurons that project to the ventral striatum. Thus, this experiment identified a synaptic correlate of impulse control. In the second experiment, a separate group of rats were trained in the RI task prior to electrophysiological testing. Training in the RI task produced a decrease in membrane excitability in prelimbic, but not infralimbic, neurons as measured by maximal spiking evoked in response to increasing current injection. Importantly, this decrease was strongly correlated with successful inhibition in the task. Fortuitously, subjects trained in an operant control condition showed elevated infralimbic, but not prelimbic, excitability, which was produced by learning an anticipatory signal that predicted imminent reward availability. These experiments revealed two cellular correlates of performance, corresponding to learning two different associations under distinct task conditions. In the final experiment, rats were trained on the RI task under three conditions: Short (4-s), long (60-s), or unpredictable (1-s to 60-s) premature phases. These conditions produced distinct errors on the RI task. Interestingly, amphetamine increased premature responding in the short and long conditions, but decreased premature responding in the unpredictable condition. This dissociation may arise from interactions between amphetamine and underlying cognitive processes, such as attention, timing, and conditioned avoidance. In summary, this thesis showed that learning to inhibit a response produces distinct synaptic, cellular, and pharmacological changes. It is hoped that these advances will provide a starting point for future therapeutic interventions of disorders of impulse control.