Postfusional control of quantal current shape

Whether glutamate is released rapidly, in an all-or-none manner, or more slowly, in a regulated manner, is a matter of debate. We analyzed the time course of excitatory postsynaptic currents (EPSCs) at glutamatergic neuromuscular junctions of Drosophila and found that the decay phase of EPSCs was protracted to a variable extent. The protraction was more pronounced in evoked and spontaneous quantal EPSCs than in action potential-evoked multiquantal EPSCs; reduced in quantal EPSCs from endophilin null mutants, which maintain release via kiss-and-run; and dependent on synaptotagmin isoform, calcium, and protein phosphorylation. Our data indicate that glutamate is released from individual synaptic vesicles for milliseconds through a fusion pore. Quantal glutamate discharge time course depends on presynaptic calcium inflow and the molecular composition of the release machinery.     

Expression mechanisms of long-term potentiation in the hippocampus

We have taken a number of different experimental approaches to address whether long-term potentiation (LTP) in hippocampal CA1 pyramidal cells is due primarily to presynaptic or postsynaptic modifications. Examination of miniature EPSCs or EPSCs evoked using minimal stimulation indicate that quantal size increasing during LTP. The conversion of silent to functional synapses may contribute to the LTP-induced changes in mEPSC frequency and failure rate that previously have been attributed to an increase in the probability if transmitter release.     

Actions of endomorphins on synaptic transmission of aδ-fibers in spinal cord dorsal horn neurons

The effects of endogenous mu-opioid ligands, endomorphins, on Adelta-afferent-evoked excitatory postsynaptic currents (EPSCs) were studied in substantia gelatinosa neurons in spinal cord slices. Under voltage-clamp conditions, endomorphins blocked the evoked EPSCs in a dose-dependent manner. To determine if the block resulted from changes in transmitter release from glutamatergic synaptic terminals, the opioid actions on miniature excitatory postsynaptic currents (mEPSCs) were examined. Endomorphins (1 microM) reduced the frequency but not the amplitude of mEPSCs, suggesting that endomorphins directly act on presynaptic terminals. The effects of endomorphins on the unitary (quantal) properties of the evoked EPSCs were also studied. Endomorphins reduced unitary content without significantly changing unitary amplitude. These results suggest that in addition to presynaptic actions on interneurons, endomorphins also inhibit evoked EPSCs by reducing transmitter release from Adelta-afferent terminals.     

Confinement Regulates Complex Biochemical Networks: Initiation of Blood Clotting by “Diffusion Acting”

This study shows that environmental confinement strongly affects the activation of nonlinear reaction networks, such as blood coagulation (clotting), by small quantities of activators. Blood coagulation is sensitive to the local concentration of soluble activators, initiating only when the activators surpass a threshold concentration, and therefore is regulated by mass transport phenomena such as flow and diffusion. Here, diffusion was limited by decreasing the size of microfluidic chambers, and it was found that microparticles carrying either the classical stimulus, tissue factor, or a bacterial stimulus, Bacillus cereus, initiated coagulation of human platelet-poor plasma only when confined. A simple analytical argument and numerical model were used to describe the mechanism for this phenomenon: confinement causes diffusible activators to accumulate locally and surpass the threshold concentration. To interpret the results, a dimensionless confinement number, Cn, was used to describe whether a stimulus was confined, and a Damköhler number, Da₂, was used to describe whether a subthreshold stimulus could initiate coagulation. In the context of initiation of coagulation by bacteria, this mechanism can be thought of as “diffusion acting”, which is distinct from “diffusion sensing”. The ability of confinement and diffusion acting to change the outcome of coagulation suggests that confinement should also regulate other biological “on” and “off” processes that are controlled by thresholds.     

5-HT4 receptor activation facilitates recovery from synaptic rundown and increases transmitter release from single varicosities of myenteric neurons

5-HT(4) receptor agonists facilitate synaptic transmission in the enteric nervous system, and these drugs are used to treat constipation. In the present study, we investigated the effects of the 5-HT(4) receptor agonist, renzapride, on rundown and recovery of fast excitatory postsynaptic potentials (fEPSPs) during and after trains of stimulation and on transmitter release from individual myenteric neuronal varicosities. Intracellular electrophysiological methods were used to record fEPSPs from neurons in longitudinal muscle myenteric plexus preparations of guinea pig ileum in vitro. During trains of supramaximal electrical stimulation (10 Hz, 2 s), fEPSP amplitude declined (time constant = 0.6 +/- 0.1 s) from 17 +/- 2 mV to 0.7 +/- 0.3 mV. Renzapride (0.1 microM) did not change the time constant for fEPSP rundown, but it decreased the time constant for recovery of fEPSP amplitude after the stimulus train from 7 +/- 2 s to 1.6 +/- 0.2 s (P < 0.05). 5-HT (0.1 microM) also increased fEPSPs and facilitated recovery from rundown. The adenylate cyclase activator, forskolin (1 muM), mimicked the actions of renzapride and 5-HT, whereas H-89, a protein kinase A (PKA) inhibitor, blocked the effects of renzapride. We used nicotinic acetylcholine receptor containing outside-out patches obtained from myenteric neurons maintained in primary culture to detect acetylcholine release from single varicosities. Renzapride (0.1 microM) increased release probability twofold. We conclude that 5-HT(4) receptors activate the adenylyl cyclase-PKA pathway to increase acetylcholine release from single varicosities and to accelerate recovery from synaptic rundown. These responses may contribute to the prokinetic actions of 5-HT(4) receptor agonists.     

Astrocyte calcium microdomains are inhibited by Bafilomycin A1 and cannot be replicated by low-level Schaffer collateral stimulation in situ

Astrocyte Gq GPCR and IP₃ receptor-dependent Ca²⁺ elevations occur spontaneously in situ and in vivo. These events vary considerably in size, often remaining confined to small territories of astrocyte processes called “microdomains” and sometimes propagating over longer distances that can include the soma. It has remained unclear whether these events are driven by constitutive (basal) GPCR signaling activity, neuronal action potential-dependent or quantal vesicular release, or some combination of these mechanisms. Here, we applied manipulations to increase or inhibit neuronal vesicular neurotransmitter release together with low-level stimulation of Schaffer collaterals in acute mouse hippocampal slices in an effort to determine the mechanisms underlying spontaneous astrocyte Ca²⁺ events. We found no significant change in spontaneous microdomain astrocyte Ca²⁺ elevations when neuronal action potentials were significantly enhanced or blocked. The astrocyte Ca²⁺ activity was also not affected by inhibitors of group I mGluRs. However, blockade of miniature neurotransmitter release using Bafilomycin A1 significantly reduced the frequency of microdomain astrocyte Ca²⁺ elevations. We then tested whether astrocyte Ca²⁺ microdomains can be evoked by low intensity SC stimulation. Importantly, microdomains could not be reproduced even using single, low intensity pulses to the SCs at a minimum distance from the astrocyte. Evoked astrocyte Ca²⁺ responses most often included the cell soma, were reduced by group I mGluR antagonists, and were larger in size compared to spontaneous Ca²⁺ microdomains. Overall, our findings suggest that spontaneous microdomain astrocyte Ca²⁺ elevations are not driven by neuronal action potentials but require quantal release of neurotransmitter which cannot be replicated by stimulation of Schaffer collaterals.     

Two reaction pathways for transformation of high potential cytochrome b559 of PS II into the intermediate potential form

This study describes an analysis of different treatments that influence the relative content and the midpoint potential of HP Cyt b559 in PS II membrane fragments from higher plants. Two basically different types of irreversible modification effects are distinguished: the HP form of Cyt b559 is either predominantly affected when the heme group is oxidized (“O-type” effects) or when it is reduced (“R-type” effects). Transformation of HP Cyt b559 to lower potential redox forms (IP and LP forms) by the “O-type” mechanism is induced by high pH and detergent treatments. In this case the effects consist of a gradual decrease in the relative content of HP Cyt b559 while its midpoint potential remains unaffected. Transformation of HP Cyt b559 via an “R-type” mechanism is caused by a number of exogenous compounds denoted L: herbicides, ADRY reagents and tetraphenylboron. These compounds are postulated to bind to the PS II complex at a quinone binding site designated as Q_C which interacts with Cyt b559 and is clearly not the Q_B site. Binding of compounds L to the Q_C site when HP Cyt b559 is oxidized gives rise to a gradual decrease in the E_m of HP Cyt b559 with increasing concentration of L (up to 10 K_ox(L) values) while the relative content of HP Cyt b559 is unaffected. Higher concentrations of compounds L required for their binding to Q_C site when HP Cyt b559 is reduced (described by K_red(L)) induce a conversion of HP Cyt b559 to lower potential redox forms (“R-type” transformation). Two reaction pathways for transitions of Cyt b559 between the different protein conformations that are responsible for the HP and IP/LP redox forms are proposed and new insights into the functional regulation of Cyt b559 via the Q_C site are discussed.     

Distinct quantal features of AMPA and NMDA synaptic currents in hippocampal neurons: implication of glutamate spillover and receptor saturation

Excitatory postsynaptic currents (EPSCs) were studied in the CA1 pyramidal cells of rat hippocampal slices. Components mediated by alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid (AMPA) and by N-methyl-D-aspartate (NMDA) receptors were separated pharmacologically. Quantal parameters of AMPA and NMDA receptor-mediated EPSCs were obtained using both maximal likelihood and autocorrelation techniques. Enhancement of transmitter release with 4-aminopyridine caused a significant increase in quantal size of NMDA EPSC. This was accompanied by a slowing of the EPSC decay. The maximal number of quanta in the NMDA current was unchanged, while the probability of quantal event dramatically enhanced. In contrast, neither the quantal size nor the kinetics of AMPA EPSC was altered by 4-aminopyridine, while the maximal number of quanta increased. These changes in the quantal parameters are consistent with a transition to multivesicular release of the neurotransmitter. Spillover of excessive glutamate on the nonsynaptic areas of dendritic spines causes an increase in the quantal size of NMDA synaptic current. The difference in quantal behavior of AMPA and NMDA EPSCs implies that different mechanisms underlie their quantization: the additive response of nonsaturated AMPA receptors contrasts with the variable involvement of saturated intrasynaptic and nonsaturated extrasynaptic NMDA receptors.     

Variability of neurotransmitter concentration and nonsaturation of postsynaptic AMPA receptors at synapses in hippocampal cultures and slices

To understand the elementary unit of synaptic communication between CNS neurons, one must know what causes the variability of quantal postsynaptic currents and whether unitary packets of transmitter saturate postsynaptic receptors. We studied single excitatory synapses between hippocampal neurons in culture. Focal glutamate application at individual postsynaptic sites evoked currents (I(glu)) with little variability compared with quantal excitatory postsynaptic currents (EPSCs). The maximal I(glu) was >2-fold larger than the median EPSC. Thus, variations in [glu]cleft are the main source of variability in EPSC size, and glutamate receptors are generally far from saturation during quantal transmission. This conclusion was verified by molecular antagonism experiments in hippocampal cultures and slices. The general lack of glutamate receptor saturation leaves room for increases in [glu]cleft as a mechanism for synaptic plasticity.     

Dynamics of mitochondrial structure during apoptosis and the enigma of Opa1

“The large scale remodeling of mitochondria during apoptosis is a necessary step for the complete release of cytochrome c” has been a tenet since 2002. However, more recent findings strongly indicate that the large-scale remodeling previously described actually takes place after the release of cytochrome c and in a caspase-dependent manner, bringing into question whether mitochondria remodeling is necessary. In a more recent article, however, it was shown that a much more subtle form of remodeling is taking place which is only observable by electron tomography. In the Bcl-2 inhibitable Bax/Bak-dependent intrinsic pathway of apoptosis, the release of cytochrome c from mitochondria is a consequence of two carefully coordinated events: formation of outer membrane pores and opening of crista junctions triggered by Opa1 oligomer disassembly, and both steps are necessary for the complete release of cytochrome c. We review the recent literature pertaining to the coordinated release of cytochrome c during cell death.     

Characterisation of physiological and immunological differences between Pacific oysters (Crassostrea gigas) genetically selected for high or low survival to summer mortalities and fed different rations under controlled conditions

Within the framework of a national scientific program named “MORtalités ESTivales de l'huître creuse Crassostrea gigas” (MOREST), a family-based experiment was developed to study the genetic basis of resistance to summer mortality in the Pacific oyster, Crassostrea gigas. As part of the MOREST project, the second generation of three resistant families and two susceptible families were chosen and pooled into two respective groups: “R” and “S”. These two groups of oysters were conditioned for 6 months on two food levels (4% and 12% of oyster soft-tissue dry weight in algal dry weight per day) with a temperature gradient that mimicked the Marennes-Oléron natural cycle during the oyster reproductive period. Oyster mortality remained low for the first two months, but then rapidly increased in July when seawater temperature reached 19 °C and above. Mortality was higher in “S” oysters than in “R” oysters, and also higher in oysters fed the 12% diet than those fed 4%, resulting in a decreasing, relative order in cumulative mortality as follows; 12% “S” > 12% “R” > 4% “S” > 4% “R”. Although the observed mortality rates were lower than those previously observed in the field, the mortality differential between “R” and “S” oysters was similar. Gonadal development, estimated by tissue lipid content, followed a relative order yielding a direct, positive relationship between reproductive effort and mortality as we reported precedently by quantitative histology. Regarding hemocyte parameters, one of the most striking observations was that reactive oxygen species (ROS) production was significantly higher in “S” oysters than in “R” oysters in May and June, regardless of food level. The absence of known environmental stress under these experimental conditions suggests that the ROS increase in “S” oyster could be related to their higher reproductive activity. Finally, a higher increase in hyalinocyte counts was observed for”S” oysters, compared to “R” oysters, in July, just before mortality. Taken together, our results suggest an association of genetically based resistance to summer mortality, reproductive strategy and hemocyte parameters.     

Merychippus (Mammalia, Perissodactyla, Equidae) from the Middle Miocene of state of Oaxaca, southeastern Mexico

Mexican material referable to Merychippus from two localities in eastern Oaxaca was described first nearly 50 years ago. Subsequent work there and in Central Oaxaca, spanning some 30 years, has allowed to establish the detail stratigraphy in both regions, and assembled a collection of merychippine material from the Matatlán (Central Oaxaca) and El Camarón (eastern Oaxaca) Formations, both K-Ar dated ~15 Ma (late early Barstovian). Detailed taxonomic analysis of this collection indicate the presence of two subhypsodont horse species referable to “Merychippus” cf. “M.” primus and “M.” cf. “M.” sejunctus in both regions. These records document the coexistence in tropical southern North America of basal and hipparionine affinity merychippine grade species, and provide a glimpse in to the diversity of subhypsodont equids in this region.     

The quantal nature of transmission and spontaneous potentials at the Torpedo electromotor junction

Miniature and stimulus evoked electroplaque potentials (mEpPs and EpPs) were recorded in Torpedo electrocytes intracellularly and extracellularly. The quantal release parameters of EpPs and the time course of quantal EpCs were estimated in normal and low Ca2+-high Mg2+ solutions. Amplitude-frequency distribution of mEpPs showed Gaussian or uneven character with an average mean value of 0.3 +/- 0.08 mV (S.D.). The mean coefficient of variation of mEpPs was 26.8 +/- 7.2% (n = 6). Tetrodotoxin reversibly blocked the stimulus evoked EpP but hardly influenced the amplitude-frequency histogram of spontaneous EpPs in 10(-8)-10(-6) M concentration. The quantum content of stimulus evoked EpPs varied between 100-400 in normal solution which decreased in low Ca2+-high Mg2+ solution and the quantal release conformed to binomial statistics and allowed determination of the parameters p and n. Frequency of the spontaneous discharges varied highly from electrocyte to electrocyte but an analysis of the time intervals showed randomness for the events. The decay phase of quantal current composed of non-exponential and exponential sections which was characteristic with 0.75 +/- 0.16 msec (mean, S.D., at 20 degrees C) time constant of exponential decay. Although, two types of mEpCs could be differentiated having significantly slower and faster time courses. Neostigmine prolonged the time constant of decay of mEpCs in dose-dependent manner with a factor of 2 in 10(-6) M and of 4 in 10(-5) M concentrations (at about 20 degrees C).     

Neurodegenerative disease-specific induced pluripotent stem cell research

Neurodegenerative disease-specific induced pluripotent stem cell (iPSC) research contributes to the following 3 areas; “Disease modeling”, “Disease material” and “Disease therapy”.“Disease modeling”, by recapitulating the disease phenotype in vitro, will reveal the pathomechanisms. Neurodegenerative disease-specific iPSC-derived non-neuronal cells harboring disease-causative protein(s), which play critical roles in neurodegeneration including motor neuron degeneration in amyotrophic lateral sclerosis, could be “Disease material”, the target cell(s) for drug screening. These differentiated cells also could be used for “Disease therapy”, an autologous cellular replacement/neuroprotection strategy, for patients with neurodegenerative disease.Further progress in these areas of research can be made for currently incurable neurodegenerative diseases.     

A random-walk interpretation of incentive effects in visual discrimination

Previous studies have shown that changes in stimulus discriminability and changes in reward density affect pigeon reaction-time (RT) distributions in different ways. A random-walk model (“RWP”) accounts for these differences and assigns a single parameter to each of the independent variables. This paper briefly reviews the model and illustrates its findings with hue discrimination data. A new analysis then presents fits to data showing that increased reward for stimulus “A” lengthens RT of pecks to an alternative stimulus “B”, and that this effect on RT distributions is much the same as the effect caused by reduction of reward to B. RWP account for both effects by changes in its “bias” parameter. The remainder of the paper comments on the relations between reward, RT, incentive and bias.     

The state transition mechanism—simply depending on light-on and -off in Spirulina platensis

The state transition in cyanobacteria is a long-discussed topic of how the photosynthetic machine regulates the excitation energy distribution in balance between the two photosystems. In the current work, whether the state transition is realized by “mobile phycobilisome (PBS)” or “energy spillover” has been clearly answered by monitoring the spectral responses of the intact cells of the cyanobacterium Spirulina platensis. Firstly, light-induced state transition depends completely on a movement of PBSs toward PSI or PSII while the redox-induced one on not only the “mobile PBS” but also an “energy spillover”. Secondly, the “energy spillover” is triggered by dissociation of PSI trimers into the monomers which specially occurs under a case from light to dark, while the PSI monomers will re-aggregate into the trimers under a case from dark to light, i.e., the PSI oligomerization is reversibly regulated by light switch on and off. Thirdly, PSI oligomerization is regulated by the local H⁺ concentration on the cytosol side of the thylakoid membranes, which in turn is regulated by light switch on and off. Fourthly, PSI oligomerization change is the only mechanism for the “energy spillover”. Thus, it can be concluded that the “mobile PBS” is a common rule for light-induced state transition while the “energy spillover” is only a special case when dark condition is involved.     

Quantal Characteristics of Synaptic GABA Release under Conditions of Stimulation of Single Synaptic Terminals of Cultured Cortical Cells of the Rat

We studied evoked inhibitory postsynaptic currents (eIPSC) using local electrical stimulation of single presynaptic terminals of cultured rat neocortical neurons. According to pharmacological and kinetic properties, these currents were qualified as GABA_A-activated. Using autocorrelation analysis of distributions of the eIPSC amplitudes, which were in all cases polymodal, we examined quantal characteristics of the above eIPSC. These results were compared with the values of quantal parameters (N, p, Q, and m) of the current families obtained using approximation by binomial distribution. Amplitude histograms of spontaneous miniature IPSC recorded under conditions of the minimum quantal release of the neurotransmitter were normal (close to Gaussian) with the mode within a 10 pA range, which is very close to analogous parameters calculated using autocorrelation and binomial techniques.     

The “Mirror” Estimate: An Intuitive Predictor of Membrane Polarization during Extracellular Stimulation

Achieving controlled extracellular microstimulation of the central nervous system requires understanding the membrane response of a neuron to an applied electric field. The “activating function” has been proposed as an intuitive predictor of membrane polarization during stimulation, but subsequent literature raised several limitations of this estimate. In this study, we show that, depending on the space constant λ, the steady-state solution to the passive cable equation is theoretically well approximated by either the activating function when λ is small, or the “mirror” image of the extracellular potential when λ is large. Using simulations, we then explore the respective domain of both estimates as a function of λ, stimulus duration, fiber length, and electrode-fiber distance. For realistic λ (>50-100 μm), the mirror estimate is the best predictor for either long electrode-fiber distances or short distances (<20-30 μm) when stimulus durations exceed a few tens of microseconds. For intermediate distances, the mirror estimate is all the more valid that the stimulus duration is long and the fiber is short. We also illustrate that this estimate correctly predicts the steady-state membrane polarization of complex central nervous system arborizations. In conclusion, the mirror estimate can often be preferred to the activating function to intuitively predict membrane polarization during extracellular stimulation.     

Stable Intermediates and Holdpoints in the Rapid Differentiation ofNaegleria

The rapidity of the optional 90-min differentiation ofNaegleria gruberifrom amoebae to flagellates suggests the possibility of a free-running cascade of events from initiating stimulus through gene expression to organelle assembly and cell morphogenesis. Instead our experiments reveal two points early in the differentiation at which the strength of the inducing stimulus is reevaluated by the cells. Two new physical start signals for differentiation, temperature downshift (ΔT) and mechanical agitation, are shown to regulate differentiation synergistically with each other and with previously defined signals. A ΔTof −10°C induces complete differentiation directly in the growth environment, whereas smaller ΔTs initiate differentiation and allow it to progress for a short time, after which the cells “hold” for up to 4 h, awaiting a stimulus to continue differentiation. Our work defines two “holdpoints,” optional points in development where progress can stop, awaiting a suitable signal, while cells retain whatever intermediates represent progress. We propose that such holdpoints, which can be detected in this system because of the temporal reproducibility of the differentiation, are likely to be found in other differentiating cells.