Models of active transport of neurotransmitters in synaptic vesicles

Models of the active transport of neurotransmitters in synaptic vesicles were constructed. The models were used to determine the resting potential at membranes of synaptic vesicles: 40mV (monoamines and acetylcholine) and -40mV (glutamate). The potential at the membrane of a synaptic vesicle was almost absent for the transport of GABA and glycine. The neurotransmitter concentration of a cell was 0.1-18mM at the concentration of neurotransmitters in a vesicle equal to 0.5M. This result is in qualitative agreement with the relevant experimental data.     

Depolarization-induced release of l-glutamic acid from isolated-resealed synaptic membrane vesicles

l-Glutamic acid actively loaded into resealed brain synaptic membrane vesicles was rapidly released into the incubation medium following the introduction of KCl and CaCl₂, or nigericin, or veratridine into the external medium. The KCl-induced release was enhanced by the presence of low (0.1 mM), extravesicular [Ca²⁺]. Neither the KCl-induced nor the veratridine-stimulated l-glutamate efflux were carrier-mediated processes. Finally, the KCl-stimulated l-glutamate efflux was dependent on the ratio of intra- to extravesicular [K⁺]. The observations described in this study were indicative of depolarization-induced l-glutamate release from isolated synaptic plasma membrane vesicles.     

l-Glutamate effects on electrical potentials of synaptic plasma membrane vesicles

The electrogenic nature of the l-glutamate-stimulated Na⁺ flux was examined by measuring the distribution of the lipophilic anion [³⁵S]thiocyanate (SCN^?) into synaptic membrane vesicles that were incubated in a NaCl medium. Concentrations of l-glutamate from 10^?7 to 10^?4 M added to the incubation medium caused an enhanced intravesicular accumulation of SCN^?. Based on the SCN^? distribution in synaptic membrane vesicles it was calculated that 10 μM l-glutamate induced an average change in the membrane potential of + 13 mV. l-Glutamate enhanced both the Na⁺ and K⁺ conductance of these membranes as determined by increases in SCN^? influx. Other neuroexcitatory amino acids and amino acid analogs (d-glutamate, l-aspartate, l-cysteine sulfinate, kainate, ibotenate, quisqualate, $\text{N-}\text{methyl}\text{-}\text{d}\text{-}\text{aspartate}$, and dl-homocysteate) also increased SCN^? accumulation in synaptic membrane vesicles. These observations are indicative of the activation by l-glutamate and some of its analogs of excitatory amino acid receptor ion channel complexes in synaptic membranes.     

Inferring network activity from synaptic noise

During intense network activity in vivo, cortical neurons are in a high-conductance state, in which the membrane potential (V(m)) is subject to a tremendous fluctuating activity. Clearly, this "synaptic noise" contains information about the activity of the network, but there are presently no methods available to extract this information. We focus here on this problem from a computational neuroscience perspective, with the aim of drawing methods to analyze experimental data. We start from models of cortical neurons, in which high-conductance states stem from the random release of thousands of excitatory and inhibitory synapses. This highly complex system can be simplified by using global synaptic conductances described by effective stochastic processes. The advantage of this approach is that one can derive analytically a number of properties from the statistics of resulting V(m) fluctuations. For example, the global excitatory and inhibitory conductances can be extracted from synaptic noise, and can be related to the mean activity of presynaptic neurons. We show here that extracting the variances of excitatory and inhibitory synaptic conductances can provide estimates of the mean temporal correlation-or level of synchrony-among thousands of neurons in the network. Thus, "probing the network" through intracellular V(m) activity is possible and constitutes a promising approach, but it will require a continuous effort combining theory, computational models and intracellular physiology.     

Dielectric noise in membranes

Most theoretical and experimental studies of electrical fluctuations in membranes so far have been devoted to noise associated with conduction processes. In this paper a different type of noise is described which results from dipolar transitions in the membrane. Two mechanisms for the generation of such dielectric noise are analyzed: (a) conformational transitions of membrane proteins involving changes in dipolar moment and/or polarizibility, and (b) rotation of dipolar molecules dissolved in the lipid. The spectral intensity of current noise calculated for the two models exhibits a characteristic dependence on frequency ω with a decrease proportional to $\text{ω}{}^2$ towards low frequencies and an approach to a frequency-independent (white noise) limit at high frequencies. For a given number of dipolar molecules in the membrane, the spectral intensity is inversely proportional to the square of the membrane thickness.     

n-Amyl alcohol partitioning in synaptic plasma membranes

Nuclear magnetic resonance and fluorescence polarization techniques were used to determine n-amyl alcohol partitioning between, and effects on, lipid microdomains of isolated rat cerebral synaptic plasma membranes, n-Amyl alcohol binding to the hydrophobic membrane core had an unchanging binding constant over an aqueous alcohol concentration range of 2.5–22.5 mM, indicating a linear relationship between membrane core and aqueous alcohol concentrations. Binding to the membrane surface, in contrast, was cooperative with a steadily increasing binding constant over this alcohol concentration range. Membrane lipid order was determined using various fluorescent probes with preferences for the membrane core, for the mid-acyl regions of the exofacial or cytofacial bilayer leaflets and for ordered or bulk microdomains. All these probes showed steady decreases in membrane order with increasing alcohol concentration, at least for the nanosecond time scale sampled by this technique. These results further demonstrate the complexity of interaction between natural membranes and membrane disordering agents.     

Generation of theta oscillations by weakly coupled neural oscillators in the presence of noise

Neuronal oscillations are a robust phenomenon occurring in a variety of brain regions despite considerable amounts of noise. In this article classical phase-response theory is generalized to the case of noisy weak-coupling regimes by deriving an iterated map for the asynchrony of spikes in an oscillation cycle. Two criteria are introduced to check the validity of our approximations: One criterion tests the assumption that all neurons fire exactly once per cycle, the other criterion tests for linearity. The framework is applied to stellate cells of the medial entorhinal cortex layer II. We find that rhythmogenesis is more robust in the case of excitatory noise as compared to inhibitory noise. It is shown that a network of stellate cells can also act as a generator of theta if the neurons are connected via a fast-oscillating network of inhibitory interneurons.     

Properties of a tonically active, sodium-permeable current in mouse urinary bladder smooth muscle

Urinary bladder smooth muscle (UBSM) elicits depolarizing action potentials, which underlie contractile events of the urinary bladder. The resting membrane potential of UBSM is approximately -40 mV and is critical for action potential generation, with hyperpolarization reducing action potential frequency. We hypothesized that a tonic, depolarizing conductance was present in UBSM, functioning to maintain the membrane potential significantly positive to the equilibrium potential for K(+) (E(K); -85 mV) and thereby facilitate action potentials. Under conditions eliminating the contribution of K(+) and voltage-dependent Ca(2+) channels, and with a clear separation of cation- and Cl(-)-selective conductances, we identified a novel background conductance (I(cat)) in mouse UBSM cells. I(cat) was mediated predominantly by the influx of Na(+), although a small inward Ca(2+) current was detectable with Ca(2+) as the sole cation in the bathing solution. Extracellular Ca(2+), Mg(2+), and Gd(3+) blocked I(cat) in a voltage-dependent manner, with K(i) values at -40 mV of 115, 133, and 1.3 microM, respectively. Although UBSM I(cat) is extensively blocked by physiological extracellular Ca(2+) and Mg(2+), a tonic, depolarizing I(cat) was detected at -40 mV. In addition, inhibition of I(cat) demonstrated a hyperpolarization of the UBSM membrane potential and decreased the amplitude of phasic contractions of isolated UBSM strips. We suggest that I(cat) contributes tonically to the depolarization of the UBSM resting membrane potential, facilitating action potential generation and thereby a maintenance of urinary bladder tone.     

Theory of single-file noise

A general theoretical approach to the analysis of electric fluctuations generated by the so-called single-file diffusion through narrow channels is presented. The formalism is a slight extension of an approach to electric fluctuations in discrete transport systems with negligible interactions between the particles recently developed by one of the authors. In the single-file transport mechanism interactions between the particles must be taken into account. Three main results of principal interest are: (a) the electric fluctuations around stationary states (at equilibrium and non-equilibrium) are determined by the time-dependent solutions of the macroscopic single-file transport equations, (b) as a direct consequence of the interactions between the ions in the single-file transport the macroscopic time-dependent current and the autocorrelation function of the microscopic current fluctuations can exhibit damped oscillatory behavior, and the current noise spectrum can show peaking, (c) the number of binding sites for the ions within the pores seems to have a strong influence on the oscillatory behavior: with increasing number of binding sites the damping of the oscillations decreases and the peaking of the spectrum becomes stronger.     

Synaptopathy under conditions of altered gravity: Changes in synaptic vesicle fusion and glutamate release

Glutamate release and synaptic vesicle heterotypic/homotypic fusion were characterized in brain synaptosomes of rats exposed to hypergravity (10 G, 1 h). Stimulated vesicular exocytosis determined as KCl-evoked fluorescence spike of pH-sensitive dye acridine orange (AO) was decreased twice in synaptosomes under hypergravity conditions as compared to control. Sets of measurements demonstrated reduced ability of synaptic vesicles to accumulate AO (10% higher steady-state baseline level of AO fluorescence). Experiments with preloaded l-[¹⁴C]glutamate exhibited similar amount of total glutamate accumulated by synaptosomes, equal concentration of ambient glutamate, but the enlarged level of cytoplasmic glutamate measuring as leakage from digitonin-permeabilized synaptosomes in hypergravity. Thus, it may be suggested that +G-induced changes in stimulated vesicular exocytosis were a result of the redistribution of intracellular pool of glutamate, i.e. a decrease in glutamate content of synaptic vesicles and an enrichment of the cytoplasmic glutamate level. To investigate the effect of hypergravity on the last step of exocytosis, i.e. membrane fusion, a cell-free system consisted of synaptic vesicles, plasma membrane vesicles, cytosolic proteins isolated from rat brain synaptosomes was used. It was found that hypergravity reduced the fusion competence of synaptic vesicles and plasma membrane vesicles, whereas synaptosomal cytosolic proteins became more active to promote membrane fusion. The total rate of homo- and heterotypic fusion reaction initiated by Ca²⁺ or Mg²⁺/ATP remained unchanged under hypergravity conditions. Thus, hypergravity could induce synaptopathy that was associated with incomplete filling of synaptic vesicles with the neuromediator and changes in exocytotic release.     

Depolarization Increases Chloride-Dependent Glutamate Sequestration in Synaptic Membranes of Rat Cerebral Cortex

To assess the functions of Cl- -dependent glutamate binding (Cl- -dependent glutamate uptake) in synaptic membranes, possible effects of depolarization on the uptake were examined. When rat cerebral cortical slices were preincubated with depolarizing agents such as veratrine (7 micrograms/ml), 10 microM aconitine, 56 mM K+, and 50 microM monensin, [3H]glutamate uptake by the crude synaptic membranes, which were subsequently prepared from the pretreated slices, was increased by 60-85%. Stimulation of the glutamate uptake by predepolarization was dependent on Na+ but not on Ca2+. The bindings of gamma-[3H]aminobutyric acid and 5-[3H]hydroxytryptamine were not significantly affected by the predepolarization. Veratrine pretreatment increased the maximal density of the glutamate uptake sites without affecting the affinity for glutamate. Several characteristics of the uptake sites increased by the veratrine pretreatment coincided with those of Cl- -dependent glutamate uptake sites. Na+-dependent glutamate binding (Na+-dependent glutamate uptake) to the membranes was not affected by pretreatment with veratrine. The content of endogenous glutamate and the noninulin space in the membrane fractions were not changed by the predepolarization. The increase in the glutamate uptake induced by pretreatment with high K+ was reversible: it returned to the control level after a second incubation of the slices in control medium. These results suggest that the Cl- -dependent glutamate sequestration system in synaptic membranes is regulated by the membrane potential.     

Analysis of the synaptic vesicle proteome using three gel-based protein separation techniques

Synaptic vesicles are key organelles in neurotransmission. Their functions are governed by a unique set of integral and peripherally associated proteins. To obtain a complete protein inventory, we immunoisolated synaptic vesicles from rat brain to high purity and performed a gel-based analysis of the synaptic vesicle proteome. Since the high hydrophobicity of integral membrane proteins hampers their resolution by gel electrophoretic techniques, we applied in parallel three different gel electrophoretic methods for protein separation prior to MS. Synaptic vesicle proteins were subjected to either 1-D SDS-PAGE along with nano-LC ESI-MS/MS or to the 2-D gel electrophoretic techniques benzyldimethyl-n-hexadecylammonium chloride (BAC)/SDS-PAGE, and double SDS (dSDS)-PAGE in combination with MALDI-TOF-MS. We demonstrate that the combination of all three methods provides a comprehensive survey of the proteinaceous inventory of the synaptic vesicle membrane compartment. The identified synaptic vesicle proteins include transporters, soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), synapsins, rab and rab-interacting proteins, additional guanine nucleotide triphosphate (GTP) binding proteins, cytoskeletal proteins, and proteins modulating synaptic vesicle exo- and endocytosis. In addition, we identified novel proteins of unknown function. Our results demonstrate that the parallel application of three different gel-based approaches in combination with mass spectrometry permits a comprehensive analysis of the synaptic vesicle proteome that is considerably more complex than previously anticipated.     

Survival-promoting activity of nimodipine and nifedipine in rat motoneurons: implications of an intrinsic calcium toxicity in motoneurons

L-type calcium channel antagonists, nimodipine and nifedipine, were tested for effects on the survival of purified rat motoneurons in culture. They showed significant activity, with maximum survival at 30 microm after 3 days in culture as high as 75%, which was comparable to the maximum effect obtained with brain-derived neurotrophic factor, a potent neurotrophic factor for rat motoneurons. It was also found that depolarizing conditions with a high potassium concentration (30 mm) were toxic to motoneurons. This toxicity was blocked by co-treatment with nimodipine. These results implicate a pre-existing calcium burden through calcium channels in motoneurons; they may offer further insights into understanding the selective death of motoneurons and have therapeutic implications in amyotrophic lateral screlosis.     

Improved stability of Drosophila larval neuromuscular preparations in haemolymph-like physiological solutions

Neuromuscular preparations from third instar larvae of Drosophila are not well-maintained in commonly used physiological solutions: vacuoles form in the muscle fibers, and membrane potential declines. These problems may result from the NaK ratio and total divalent cation content of these physiological solutions being quite different from those of haemolymph. Accordingly haemolymph-like solutions, based upon ion measurements of major cations, were developed and tested. Haemolymph-like solutions maintained the membrane potential at a relatively constant level, and prolonged the physiological life of the preparations. Synaptic transmission was well-maintained in haemolymph-like solutions, but the excitatory synaptic potentials had a slower time course and summated more effectively with repetitive stimulation, than in standard Drosophila solutions. Voltage-clamp experiments suggest that these effects are linked to more pronounced activation of muscle fiber membrane conductances in standard solutions, rather than to differences in passive muscle membrane properties or changes in postsynaptic receptor channel kinetics. Calcium dependence of transmitter release was steep in both standard and haemolymph-like solutions, but higher external calcium concentrations were required for a given level of release in haemolymph-like solutions. Thus, haemolymph-like solutions allow for prolonged, stable recording of synaptic transmission.Abbreviations HL haemolymph-like     

Glycoprotein Properties of Muscarinic Acetylcholine Receptors from Bovine Cerebral Cortex

Muscarinic acetylcholine receptors from bovine cerebral cortex were solubilized in digitonin for the subsequent determination of several biochemical properties. The digitonin-solubilized receptors were representative of the entire membrane-bound population of muscarinic receptors with respect to carbohydrate content, isoelectric point, and molecular weight. The glycoprotein nature of the solubilized receptors was demonstrated by their quantitative binding to wheat germ agglutinin-agarose. The presence of a bound antagonist did not decrease the extent of receptor binding to this lectin. Treatment of receptors with neuraminidase to remove N-acetylneuraminic acid residues reduced binding to wheat germ agglutinin-agarose by 40%; further treatment with endoglycosidases D and H, to remove all N-linked carbohydrate, decreased binding by a total of 67%. Removal of N-acetylneuraminic acid residues had no effect on agonist binding properties of the membrane-bound receptors. The carbohydrate-specific enzymes were further used to assess the contribution of carbohydrate to the isoelectric point and molecular weight of the receptor. Muscarinic receptors solubilized in either digitonin or Triton X-100 focused as one major species with a pI of 4.3. Neuraminidase treatment resulted in an increase of 0.17 units in the pI of the receptor. Muscarinic receptors labeled with the covalent muscarinic antagonist propylbenzilylcholine mustard migrated as a single major polypeptide with a molecular weight of 73,000 on sodium dodecyl sulfate-urea-polyacrylamide gels. The exclusion of urea from these gels severely retarded receptor mobility, indicating a strong tendency for aggregation of receptors in SDS. Removal of N-linked carbohydrate by endoglycosidase treatment reduced the molecular weight of the antagonist binding polypeptide by no more than 5%. These results demonstrate the glycoprotein nature of muscarinic receptors from mammalian cerebral cortex and provide evidence for their heterogeneity with respect to carbohydrate content.     

Characterization of DIDS-sensitive ATP accumulation in brain synaptic vesicles

ATP is an excitatory neurotransmitter in the central and peripheral nervous system. We investigated ATP accumulation in highly purified brain synaptic vesicles (SVs). Based on the amount of ATP accumulated in SVs under the conditions used, ATP is not transported against a concentration gradient but rather appears to have a Delta mu H(+)-independent mechanism. ATP transport was inhibited by DIDS and NEM, but was not affected by Mg(2+) or by pre-incubation with nucleotides. These results suggest a unique transport mechanism that does not involve exchange with other nucleotides or protons, unlike other known neurotransmitter transport systems.     

大鼠坐骨神经切断后外源性bcl-2对脊髓运动神经元的保护作用

采用大鼠坐骨神经切断损伤模型,行神经外膜端端对线缝合,术中依不同组别,动物于神经缝合处远端0．5cm处分别注射人的正义和反义bcl-2重组腺病毒(Ad／s-bcl-2、Ad／as-bcl-2),报道基因重组腺病毒(Ad／lacZ)和生理盐水。术后48h,7d,15d和30d常规灌注固定大鼠,取L4-L6脊髓节段,应用X-gal染色、bel-2原位杂交和免疫组化染色、TUNEL染色以及乙酰胆碱酯酶(AChE)组织化学染色方法,观察到外源基因能在脊髓中表达,同时外源性Ad／s-bcl-2能显著减少L4到L6节段脊髓前角运动神经元凋亡的数目,减少脊髓前角运动神经元中因坐骨神经切断导致的AChE活性的降低幅度,并加快其恢复。而Ad／as-bcl-2可显著增加坐骨神经切断诱导的脊髓前角运动神经元凋亡数目以及AChE活性降低幅度,并延缓其恢复。这些观察结果表明,外源性bcl-2能保护周围神经切断后引起的脊髓运动神经元损伤。     

Ultracytochemistry of the synaptic ribbons in the rat pineal organ

Summary The synaptic complexes of the rat pinealocytes are neither cholinergic nor adrenergic. In the synaptic vesicles, a neurotransmitter carrier substance of lipid nature reacting with OsO₄-Zn I₂ mixture (similar to that present in both cholinergic and adrenergic vesicles) was not found.In addition, there were no indications of glucose-6-phosphatase or thiamine-pyrophosphatase activity in the synaptic vesicles. Thus, it appears that the synaptic vesicles do not originate from the rough or smooth endoplasmic reticulum.The synaptic ribbons do not contain carbohydrates, are of protein nature and possess some chemical resemblance to microtubules and microtubular bouquets.Appropriate ultracytochemical reactions have not shown detectable quantities of sodium and calcium ions in pinealocyte synaptic complexes.Grateful acknowledgment is made to Mr. P.-A. Milliquet for technical assistance and to Dr. T. Jalanti (C.M.E., Lausanne) for his help in the use of the X-ray microanalyser.Dedicated to Professor Dr. med. G. Töndury on the occasion of his 70th birthday.     

Circadian changes in synaptic ribbons and spherules in pinealocytes of the Syrian hamster (Mesocricetus auratus)

Summary In the present study, synaptic ribbons were studied morphologically and quantitatively in hamster pineal gland. The number of ribbons and spherules of hamster pinealocytes was counted over a 24-h period. The 24-h variations in the quantity of synaptic ribbons were found to parallel fluctuations in pineal melatonin concentrations. No significant circadian changes were observed for synaptic spherules, indicating different roles for these two structures.     

A computational study of synaptic mechanisms of partial memory transfer in cerebellar vestibulo-ocular-reflex learning

There is a debate regarding whether motor memory is stored in the cerebellar cortex, or the cerebellar nuclei, or both. Memory may be acquired in the cortex and then be transferred to the cerebellar nuclei. Based on a dynamical system modeling with a minimal set of variables, we theoretically investigated possible mechanisms of memory transfer and consolidation in the context of vestibulo-ocular reflex learning. We tested different plasticity rules for synapses in the cerebellar nuclei and took robustness of behavior against parameter variation as the criterion of plausibility of a model variant. In the most plausible scenarios, mossy-fiber nucleus-neuron synapses or Purkinje-cell nucleus-neuron synapses are plastic on a slow time scale and store permanent memory, whose content is passed from the cerebellar cortex storing transient memory. In these scenarios, synaptic strengths are potentiated when the mossy-fiber afferents to the nuclei are active during a pause in Purkinje-cell activities. Furthermore, assuming that mossy fibers create a limited variety of signals compared to parallel fibers, our model shows partial memory transfer from the cortex to the nuclei.