Phase-transition-driven synaptic exocytosis: a hypothesis and its physiological and evolutionary implications

It is proposed that the plasma membrane in the active zones of synaptic terminals contains self-assembling cooperative domains whose Ca2+-induced solidification may be the driving force of the fast neurotransmitter release in the central synapses. This hypothesis and a qualitative model of the phase-transition-driven exocytosis provide formulation of a unitary approach to a number of general problems in the physiology of animals. It allows answering the following questions, among others: (i) What is the physical reason for the existence of a narrow optimum range of body temperatures in warm-blooded species? (ii) What is the physical reason for the inevitable necessity of regular sleep in animals? (iii) Does there indeed exist any general mechanism of general anesthesia?     

Calcium dependent neurotransmitter release and protein phosphorylation in synaptic vesicles.

A highly purified preparation of synaptic vesicles was prepared to study the relationship between calcium-dependent neurotransmitter release and protein phosphorylation. Calcium ions simultaneously produced significant increases in both the endogenous release of norepinephrine from the synaptic vesicles and the endogenous incorporation of [³²p] phosphate into specific synaptic vesicle proteins. The results are compatible with the hypothesis that the action of calcium on the phosphorylation of specific synaptic vesicle proteins is the molecular mechanism mediating some of the effects of calcium on neurotransmitter release and synaptic vesicle function.     

The chemical bases of the various AIDS epidemics: Recreational drugs,anti-viral chemotherapy and malnutrition

In 1981 a new epidemic of about two-dozen heterogeneous diseases began to strike non-randomly growing numbers of male homosexuals and mostly male intravenous drug users in the US and Europe. Assuming immunodeficiency as the common denominator the US Centers for Disease Control (CDC) termed the epidemic, AIDS, for acquired immunodeficiency syndrome. From 1981-1984 leading researchers including those from the CDC proposed that recreational drug use was the cause of AIDS, because of exact correlations and of drug-specific diseases. However, in 1984 US government researchers proposed that a virus, now termed human immunodeficiency virus (HIV), is the cause of the non-random epidemics of the US and Europe but also of a new, sexually random epidemic in Africa. The virus-AIDS hypothesis was instantly accepted, but it is burdened with numerous paradoxes, none of which could be resolved by 2003: Why is there no HIV in most AIDS patients, only antibodies against it? Why would HIV take 10 years from infection to AIDS? Why is AIDS not self-limiting via antiviral immunity? Why is there no vaccine against AIDS? Why is AIDS in the US and Europe not random like other viral epidemics? Why did AIDS not rise and then decline exponentially owing to antiviral immunity like all other viral epidemics? Why is AIDS not contagious? Why would only HIV carriers get AIDS who use either recreational or anti-HIV drugs or are subject to malnutrition? Why is the mortality of HIV-antibody-positives treated with anti-HIV drugs 7–9%, but that of all (mostly untreated) HIV-positives globally is only 1–4%? Here we propose that AIDS is a collection of chemical epidemics, caused by recreational drugs, anti-HIV drugs, and malnutrition. According to this hypothesis AIDS is not contagious, not immunogenic, not treatable by vaccines or antiviral drugs, and HIV is just a passenger virus. The hypothesis explains why AIDS epidemics strike non-randomly if caused by drugs and randomly if caused by malnutrition, why they manifest in drug- and malnutrition-specific diseases, and why they are not self-limiting via anti-viral immunity. The hypothesis predicts AIDS prevention by adequate nutrition and abstaining from drugs, and even cures by treating AIDS diseases with proven medications.     

Role of temperature in quanta mechanisms of facilitation in the frog neuromuscular junction

The results of computer simulations on the Double Barrier Synapse (DBS) model are presented which quantify the relationship between the synapse parameters and the quanta transfer process. The DBS model is applicable to a variety of states of synaptic activity, and by changing the synapse parameters it is possible to simulate various conditions of quanta transmission. The influence of the bathing solution temperature change on the synaptic parameters under different conditions of transmitter release in the frog neuromuscular junction is investigated. Simulations demonstrate that several synaptic parameters, including the parameters of the presynaptic membrane, are not affected by the temperature change. It is shown that a stimulation frequency exists at which the steady-state level of facilitation during a long train of stimuli is the same for a wide range of temperatures. Received: 2 August 1996 / Accepted in revised form: 19 February 1998     

Electron-microscope cytochemistry of acetylcholine-like cation by means of low-temperature "ionic fixation"

A fresh preparation of frog neuromuscle was fixed at low temperatures (0 degree-4 degrees C) by means of an "ionic-fixation" procedure which is based on the precipitation of quaternary ammonium cations, such as choline and acetylcholine, with molybdic or tungstic heteropolyanions. A low temperature was used to slow down drastically the biological processus of vesicular exocytosis so that ionic fixation, the speed of which is only slightly influenced by temperature variation, could be performed efficiently. In addition to the conventional point-like precipitate in the synaptic vesicle which is considered to be vesicular acetylcholine, numerous spot-like precipitates were observed in the synaptic cleft. Most of these were contiguous to the active zone, and some were in a paired form and corresponded to the double rows of the synaptic vesicles in contact with active zones. It is concluded that these spot-like precipitates were acetylcholine-like cations of the synaptic vesicles which had been discharged into the synaptic cleft by exocytosis and captured by the ionic fixation procedure. The results are discussed in relation to the vesicular and non-vesicular hypothesis of acetylcholine release.     

The calmodulin hypothesis of neurotransmission

Ca²⁺ plays a major role in neurotransmission and synaptic modulation. Evidence is presented to support the calmodulin hypothesis of neurotransmission developed in this laboratory stating that calmodulin, a major Ca²⁺ binding protein in brain, mediates the effects of Ca²⁺ on neurotransmission. Calmodulin was isolated from highly enriched preparations of synaptic vesicles and nerve terminal cytoplasm. Ca²⁺ and calmodulin were shown to regulate several synaptic processes in isolated and intact preparations, including endogenous synaptic Ca²⁺-calmodulin protein kinase activity, neurotransmitter release, and synaptic vesicle and synaptic membrane interactions. Ca²⁺ and calmodulin were shown to activate a synaptic tubulin kinase system which was shown to be a distinct enzyme system from the cyclic AMP protein kinase. Ca²⁺ and calmodulin stimulated phosphorylation of tubulin altered the properties of tubulin, forming insoluble tubulin fibrils. Evidence for the role of Ca²⁺-calmodulin kinase activity, especially the calmodulin-tubulin kinase, in neurotransmission are presented. The effects of several neuroactive drugs on the synaptic calmodulin system are presented. The results support the hypothesis that calmodulin mediates many of calcium's actions at the synapse, and that the effects of Ca²⁺ on synaptic protein phosphorylation, especially synaptic tubulin, may provide a biochemical mechanism for converting the Ca²⁺ signal into a motor force in the process of neurotransmission.     

Force generation and temperature-jump and length-jump tension transients in muscle fibers.

Muscle tension rises with increasing temperature. The kinetics that govern the tension rise of maximally Ca(2+)-activated, skinned rabbit psoas fibers over a temperature range of 0-30 degrees C was characterized in laser temperature-jump experiments. The kinetic response is simple and can be readily interpreted in terms of a basic three-step mechanism of contraction, which includes a temperature-sensitive rapid preequilibrium(a) linked to a temperature-insensitive rate-limiting step and followed by a temperature-sensitive tension-generating step. These data and mechanism are compared and contrasted with the more complex length-jump Huxley-Simmons phases in which all states that generate tension or bear tension are perturbed. The rate of the Huxley-Simmons phase 4 is temperature sensitive at low temperatures but plateaus at high temperatures, indicating a change in rate-limiting step from a temperature-sensitive (phase 4a) to a temperature-insensitive reaction (phase 4b); the latter appears to correlate with the slow, temperature-insensitive temperature-jump relaxation. Phase 3 is absent in the temperature-jump, which excludes it from tension generation. We confirm that de novo tension generation occurs as an order-disorder transition during phase 2slow and the equivalent, temperature-sensitive temperature-jump relaxation.     

d-Serine: A key to synaptic plasticity?

Two discoveries have put d-serine in the spotlight of neuroscience. First, d-serine was detected in brain tissue at high levels. Second, it was found to act on the N-methyl-d-aspartate receptor (NMDAR). This receptor is central to use-dependent synaptic plasticity, the cellular process which is widely believed to underlie learning. The ensuing quest for the mechanisms of d-serine synthesis, release and clearance, as well as for its physiological significance has provided a wealth of experimental evidence implicating d-serine in synaptic plasticity. However some key questions remain unanswered. Which cells release d-serine and upon what stimuli? Is d-serine supply dynamically regulated? What is the fate of released d-serine? Answering these questions appears to be an essential step in our understanding of how NMDARs trigger synaptic plasticity and learning. This review will highlight some recent advances and avenues of enquiry in dynamic d-serine signaling in the mammalian brain with emphasis on neurophysiology.     

Rate of quantal transmitter release at the mammalian rod synapse.

Under scotopic conditions, the mammalian rod encodes either one photon or none within its integration time. Consequently the signal presented to its synaptic terminal is binary. The synapse has a single active zone that releases neurotransmitter quanta tonically in darkness and pauses briefly in response to a rhodopsin isomerization by a photon. We asked: what minimum tonic rate would allow the postsynaptic bipolar cell to distinguish this pause from an extra-long interval between quanta due to the stochastic timing of release? The answer required a model of the circuit that included the rod convergence onto the bipolar cell and the bipolar cell''s signal-to-noise ratio. Calculations from the model suggest that tonic release must be at least 40 quanta/s. This tonic rate is much higher than at conventional synapses where reliability is achieved by employing multiple active zones. The rod''s synaptic mechanism makes efficient use of space, which in the retina is at a premium.     

Are exocytosis mechanisms neurotransmitter specific?

Neurotransmission is a multistage regulated process in which a variety of active molecules contained in vesicles are liberated in response to specific stimuli from different types of neurone or related cells. This includes the release of fast neurotransmitters such as amino acids and acetylcholine from central and peripheral synapses, but also that of relatively slow-acting polypeptides from central and peripheral neurones or neuroendocrine cells. Considerable progress has been made over recent years in the understanding at a molecular level of the mechanism of regulated exocytosis, a crucial phase in this phenomenon. The currently proposed overall mechanism, which incorporates the “SNARE” hypothesis for vesicle-membrane docking and fusion, is based on data from experimental models ranging from brain synaptosomes to mast cells. Since the kinetics of the models studied and the physiological effects of the neurotransmitters implicated vary so much, it is pertinent to question whether a general mechanism can be proposed from such experimental data. This review examines known differences in putative exocytotic mechanisms for the various systems studied and attempts to relate these to the nature of the active substances released. Differences exist in each step of the exocytosis process and include the channel through which Ca²⁺ enters to trigger it or the internal Ca²⁺ source, the type of vesicle in which the transmitter is packaged, the way vesicles are translocated to the surface membrane or how they dock and fuse with it. Major differences have been reported in release mechanisms of different types of vesicle, but minor differences also exist within the same vesicle class. Thus small synaptic vesicles and large dense core vesicles are translocated by distinct processes and the Ca²⁺ channels, Ca²⁺ sensors and docking proteins involved in other steps are not identical in all neuronal phenotypes. It may be concluded that each of these differences has evolved to accommodate the different physiological requirements of the neuromodulator released.     

Neurotransmitter release: fusion or 'kiss-and-run'?

The clear synaptic vesicles of neurons release their contents at the presynaptic membrane and are then quickly retrieved. However, it is unclear whether a complete cycle of exocytosis and endocytosis is always involved or whether neurotransmitter can be released by a transient interaction. Recent findings in chromaffin and mast cells suggest that exocytosis is preceded by the formation of a pore that has similar conductance properties to ion channels. The content of the secretory organelle partially escapes at this early step, but the pore can close before the vesicle fuses fully. This article looks at the evidence that quantal release of neurotransmitter from clear synaptic vesicles may occur by a similar 'kiss-and-run' mechanism.     

One-vesicle hypothesis for neurotransmitter release: A possible molecular mechanism

Neurotransmitter-containing vesicles are clustered in release sites. Although a given site can contain tens of vesicles, there is evidence that under a wide range of conditions, following an action potential, rarely is more than one vesicle released from each site. Such findings led to the one vesicle hypothesis, for which this paper suggests a molecular mechanism. The release of a vesicle from a site provides a transient high concentration of transmitter in that site. It is proposed here that the local high transmitter concentration interrupts further vesicle releases from the same release site. The suggested mechanism for this ‘release interruption’ is based on a theory of release control by the authors wherein inhibitory transmitter autoreceptors play a central role. (That transmitter binding to these autoreceptors can inhibit release on a fast time scale has recently been shown experimentally.) A detailed kinetic scheme is presented for the proposed mechanism. Stochastic simulations of this scheme demonstrate how the mechanism accounts for the one vesicle hypothesis. In agreement with recent experiments, the simulations also show that changes in conditions that affect the release process can cause frequent release of more than one vesicle per site.     

Nonsynaptic and nonvesicular ATP release from neurons and relevance to neuron-glia signaling

Studies on the release of ATP from neurons began with the earliest investigations of quantal neurotransmitter release in the 1950s, but in contrast to ATP release from other cells, studies of ATP release from neurons have been narrowly constrained to one mechanism, vesicular release. This is a consequence of the prominence of synaptic transmission in neuronal communication, but nonvesicular mechanisms for ATP release from neurons are likely to have a broader range of functions than synaptic release. Investigations of activity-dependent communication between axons and myelinating glia have stimulated a search for mechanisms that could release ATP from axons and other nonsynaptic regions in response to action potential firing. This has identified volume-activated anion channels as an important mechanism in activity-dependent ATP release from axons, and renewed interest in micromechanical changes in axons that accompany action potential firing.     

Development and optimization of solid lipid nanoparticles of amikacin by central composite design

Solid lipid nanoparticles (SLNs) have been studied as a drug-delivery system for the controlling of drug release. These colloidal systems have many important advantages, such as biocompatibility, good tolerability, and ease of scale-up. In the preparation of SLNs, many factors are involved in the characteristics of the particles, such as particle size, drug loading, and zeta potential. In this study, fractional factorial design was applied to examine which variables affect the physicochemical properties of amikacin SLNs. Study was continued by a statistical central composite design (CCD) to minimize particle size and maximize drug-loading efficiency of particles. The results showed that three quantitative factors, including the amount of lipid phase, ratio of drug to lipid, and volume of aqueous phase, were the most important variables on studied responses. The best predicted model for particle size was the quadratic model, and for drug-loading efficiency, was the linear model without any significant lack of fit. Optimum condition was achieved when the ratio of drug to lipid was set at 0.5, the amount of lipid phase at 314?mg, and the volume of aqueous phase at 229?mL. The optimized particle size was 149?±?4?nm and the drug-loading efficiency 88?±?5%. Polydispersity index was less than 0.3. The prepared particles had spherical shape, and the drug release from nanoparticles continued for 144 hours (6 days) without significant burst effect.     

The biophysical basis underlying the maintenance of early phase long-term potentiation

The maintenance of synaptic changes resulting from long-term potentiation (LTP) is essential for brain function such as memory and learning. Different LTP phases have been associated with diverse molecular processes and pathways, and the molecular underpinnings of LTP on the short, as well as long time scales, are well established. However, the principles on the intermediate time scale of 1-6 hours that mediate the early phase of LTP (E-LTP) remain elusive. We hypothesize that the interplay between specific features of postsynaptic receptor trafficking is responsible for sustaining synaptic changes during this LTP phase. We test this hypothesis by formalizing a biophysical model that integrates several experimentally-motivated mechanisms. The model captures a wide range of experimental findings and predicts that synaptic changes are preserved for hours when the receptor dynamics are shaped by the interplay of structural changes of the spine in conjunction with increased trafficking from recycling endosomes and the cooperative binding of receptors. Furthermore, our model provides several predictions to verify our findings experimentally.     

Shear stress induced lipid order and permeability changes of giant unilamellar vesicles

BackgroundThe permeability of a lipid bilayer is a function of its phase state and depends non-linearly on thermodynamic variables such as temperature, pressure or pH. We investigated how shear forces influence the phase state of giant unilamellar vesicles and their membrane permeability.MethodsWe determined the permeability of giant unilamellar vesicles composed of different phospholipid species under shear flow in a tube at various temperatures around and far off the melting point by analyzing the release of fluorescently labelled dextran. Furthermore, we quantified phase state changes of these vesicles under shear forces using spectral decomposition of the membrane embedded fluorescent dye Laurdan.ResultsWe observed that the membrane permeability follows a step function with increasing permeability at the transition from the gel to the fluid phase and vice versa. Second, there was an all-or-nothing permeabilization near the main phase transition temperature and a gradual dye release far off the melting transition. Third, the Laurdan phase state analysis suggests that shear forces induce a reversible melting temperature shift in giant unilamellar vesicle membranes.Major conclusionsThe observed effects can be explained best in a scenario in which shear forces directly induce membrane pores that possess relatively long pore lifetimes in proximity to the phase transition.General significanceOur study elucidates the release mechanism of thermo-responsive drug carriers as we found that liposome permeabilization is not continuous but quantized. Furthermore, the shear force induced melting temperature shift must be taken into consideration when thermo-responsive liposomes are designed.     

Effect of the permeability of lysosomal and mitochondrial membranes on the ability of their cryoextracts to inhibit the protein-synthesizing activity of cell-free systems

Changes in permeability of lysosomal and mitochondrial membranes were studied under different temperatures at which cryoextracts from organelles inhibit most distinctly the protein synthesizing activity of the cell-free system from the rat liver. It is found that mitochondria are more sensitive to the effect of low temperature effect than lysosomes. Overcooling of the mitochondria suspension to the temperature of the free water crystallization is shown to cause no release of the protein synthesis inhibitor. The inhibitor release from organelles occurs from the moment of crystallization and reaches its maximum at the eutectic temperatures of the freezing medium which is due to the injury effect of the complex of physicochemical factors on the membrane structures, occurring during the phase transition of the solvent.     

Reproductive mode may determine geographic distributions in Australian venomous snakes (Pseudechis,Elapidae)

Summary Why are viviparous squamate reptiles more common in cold climates, and oviparous ones in warmer areas? The usual explanation is that (1) oviparous squamates cannot reproduce successfully in cold areas because soil temperatures are too low for embryonic development; and (2) viviparous squamates experience lower survivorship or reproductive success than oviparous taxa in warmer areas. These hypotheses suggest that the boundaries of geographic distributions of congeneric oviparous and viviparous squamates should be predictable from data on thermal tolerances of embryos, and estimated temperatures of soils and gravid female reptiles throughout the potential geographic range of the taxon. In large venomous Australian snakes of the genus Pseudechis, distributional boundaries of oviparous and viviparous taxa can be accurately predicted from such data. This predictive ability, if substantiated by studies of other reproductively biomodal squamate taxa, would support the putative role of reproductive mode as a direct determinant of reptilian geographic distributions.