Is replenishment of the readily releasable pool associated with vesicular movement?

At the excitatory synapse of rat hippocampus the short-term synaptic depression observed during long high-frequency stimulation is associated with slower replenishment of the readily-releasable pool. Given that the replenishment rate is also not [Ca⁺⁺]_o sensitive this puts into question a widely held notion that the vesicles—constrained by the cytoskeleton and rendered free from such constraints by Ca⁺⁺ entry that renders them more mobile—are important in the replenishment of the readily-releasable pool. This raises a question—Is vesicular replenishment of the readily releasable pool associated with significant movement? To answer this question we evaluated how okadaic acid and staurosporine (compounds known to affect vesicular mobility) influence the replenishment rate. We used patterned stimulation on the Schaffer collateral fiber pathway and recorded the excitatory post-synaptic currents (EPSCs) from rat CA1 neurons, in the absence and presence of these drugs. The parameters of a circuit model with two vesicular pools were estimated by minimizing the squared difference between the ESPC amplitudes and simulated model output. [Ca²⁺]_o did not influence the progressive decrease of the replenishment rate during long, high frequency stimulation. Okadaic acid did not significantly affect any parameters of the vesicular storage and release system, including the replenishment rate. Staurosporine reduced the replenishment coupling, but not the replenishment rate, and this is owing to the fact that it also reduces the ability of the readily releasable pool to contain quanta. Moreover, these compounds were ineffective in influencing how the replenishment rate decreases during long, high frequency stimulation. In conclusion at the excitatory synapses of rat hippocampus the replenishment of the readily releasable pool does not appear to be associated with a significant vesicular movement, and during long high frequency stimulation [Ca⁺⁺]_o does not influence the progressive decrease of vesicular replenishment.     

Synaptic activity slows vesicular replenishment at excitatory synapses of rat hippocampus

Short-term synaptic depression mainly reflects the depletion of the readily releasable pool (RRP) of quanta. Its dynamics, and especially the replenishment rate of the RRP, are still not well characterized in spite of decades of investigation. Main reason is that the vesicular storage and release system is treated as time-independent. If it is time-dependent all parameters thus estimated become problematic. Indeed the reports about how prolonged stimulation affects the dynamics are contradictory. To study this, we used patterned stimulation on the Schaeffer collateral fiber pathway and model-fitting of the excitatory post-synaptic currents (EPSC) recorded from CA1 neurons in rat hippocampal slices. The parameters of a vesicular storage and release model with two pools were estimated by minimizing the squared difference between the ESPC amplitudes and simulated model output. This yields the ‘basic’ parameters (release coupling, replenishment coupling and RRP size) that underlie the ‘derived’ and commonly used parameters (fractional release and replenishment rate). The fractional release increases when [Ca⁺⁺]_o is raised, whereas the replenishment rate is [Ca⁺⁺]_o independent. Fractional release rises because release coupling increases, and the RRP becomes less able to contain quanta. During prolonged stimulation, the fractional release remains generally unaltered, whereas the replenishment rate decreases down to ~10 % of its initial value with a decay time of ~15 s, and this decrease in the replenishment rate significantly contributes to synaptic depression. In conclusion, the fractional release is [Ca⁺⁺]_o-dependent and stimulation-independent, whereas the replenishment rate is [Ca⁺⁺]_o-independent and stimulation-dependent.     

Temperature dependence of vesicular dynamics at excitatory synapses of rat hippocampus

How vesicular dynamics parameters depend on temperature and how temperature affects the parameter change during prolonged high frequency stimulation was determined by fitting a model of vesicular storage and release to the amplitudes of the excitatory post-synaptic currents (EPSC) recorded from CA1 neurons in rat hippocampal slices. The temperature ranged from low (13 °C) to higher and more physiological temperature (34 °C). Fitting the model of vesicular storage and release to the EPSC amplitudes during a single pair of brief high–low frequency stimulation trains yields the estimates of all parameters of the vesicular dynamics, and with good precision. Both fractional release and replenishment rate decrease as the temperature rises. Change of the underlying ‘basic’ parameters (release coupling, replenishment coupling and readily releasable pool size), which the model-fitting also yields is complex. The replenishment coupling between the readily releasable pool (RRP) and resting pool increases with temperature (which renders the replenishment rate higher), but this is more than counterbalanced by greater RRP size (which renders the replenishment rate lower). Finally, during long, high frequency patterned stimulation that leads to significant synaptic depression, the replenishment rate decreases markedly and rapidly at low temperatures (<22 °C), but at high temperatures (>28 °C) the replenishment rate rises with stimulation, making synapses better able to maintain synaptic efficacy.     

Mathematical theory of chemical synaptic transmission

Mathematical theory of chemical synaptic transmission is suggested in which the modes of operation of chemical synapses are given as consequencies of some fundamental theoretical principles presented in the form of systems of quantum and macroscopic postulates. These postulates establish transmitter transfer rules between 3 component parts — cytoplasmic, vesicular and external pools of neurotransmitter. The main features of the transfers are determined by special properties of the dividing membranes (synaptic and vesicle) which show high selectivity towards the direction of the transmitter quantum transfer. The formulation of a previously unknown effect of transmitter quantum transfer from the vesicular pool into the cytoplasmic one is introduced: it is postulated that each arriving presynaptic impulse not only releases a constant fraction of the current contents of the cytoplasmic pool into the synaptic cleft (external pool), but also realizes practically simultaneous transmitter transfer from the vesicular pool into the cytoplasmic one. Zone structure of the vesicular pool is postulated. In accordance with basic equations of the theory a nonlinear control system (dynamic synaptic modulator — DYSYM) of transmitter release from the terminal is constructed.Depending on the parameters relation two types of synapses are classified — those with rapid and slow demobilization. Analytical dependencies of the transmitter pools sizes on the stimulation frequency are introduced. By fitting the frequency dependencies to the empirical data model parameters are determined corresponding to a set of experimentally studied synaptic junctions. Different aspects of the chemical synapse behaviour under the influence of presynaptic stimulation are simulated.     

Synthesis,subcellular distribution and turnover of dopamine beta-hydroxylase in organ cultures of sympathetic ganglia and adrenal medullae

The synthesis, subcellular distribution and turnover of dopamine beta-hydroxylase was studied in organ cultures of rat adrenal medullae and superior cervical ganglia. After exposure to [3H]leucine for 1 or 3 h, the tissues were homogenized at various time intervals and the amount of labelled dopamine beta-hydroxylase in different subcellular fractions (cytosol, soluble and membrane-bound fraction of catecholamine storage vesicles) was determined by immunoprecipitation and subsequent electrophoresis. In cultured adrenal medullae, induction of dopamine beta-hydroxylase initiated in vivo by administration of reserpine affected both soluble and membrane-bound pools of dopamine beta-hydroxylase to a similar extent after pulse-labelling for 1 or 3 h. The half-lives of dopamine beta-hydroxylase, which amounted to 6 h for the cytosol, 7.5 h for the soluble vesicular and 32 h for the membrane-bound vesicular pools were not altered by pretreatment with reserpine. In superior cervical ganglia the half-lives of the soluble pools were 2-3 times longer than in the adrenal medulla, whereas the half-life of the membrane-bound fraction was the same as in the adrenal medulla. In both organs the most heavily labelled fraction (both after a pulse of 1 or 3 h) was always that of the vesicular membrane, suggesting that newly-synthesized dopamine beta-hydroxylase is immediately incorporated into the storage vesicles and not via release into the cytosol from the site of synthesis. The fact that the half-life of membrane-bound dopamine beta-hydroxylase is markedly longer than that of the two soluble pools suggests that the single pools are not only independently supplied by newly-synthesized DBH but there is also no appreciable subsequent exchange between soluble and membrane-bound pools.     

Quantitative Analysis of Synaptic Vesicle Pool Replenishment in Cultured Cerebellar Granule Neurons using FM Dyes

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis. Retrieved SVs are then refilled with neurotransmitter and rejoin the recycling pool, defined as SVs that are available for exocytosis^1,2. The recycling pool can generally be subdivided into two distinct pools - the readily releasable pool (RRP) and the reserve pool (RP). As their names imply, the RRP consists of SVs that are immediately available for fusion while RP SVs are released only during intense stimulation^1,2. It is important to have a reliable assay that reports the differential replenishment of these SV pools in order to understand 1) how SVs traffic after different modes of endocytosis (such as clathrin-dependent endocytosis and activity-dependent bulk endocytosis) and 2) the mechanisms controlling the mobilisation of both the RRP and RP in response to different stimuli.FM dyes are routinely employed to quantitatively report SV turnover in central nerve terminals^3-8. They have a hydrophobic hydrocarbon tail that allows reversible partitioning in the lipid bilayer, and a hydrophilic head group that blocks passage across membranes. The dyes have little fluorescence in aqueous solution, but their quantum yield increases dramatically when partitioned in membrane⁹. Thus FM dyes are ideal fluorescent probes for tracking actively recycling SVs. The standard protocol for use of FM dye is as follows. First they are applied to neurons and are taken up during endocytosis (Figure 1). After non-internalised dye is washed away from the plasma membrane, recycled SVs redistribute within the recycling pool. These SVs are then depleted using unloading stimuli (Figure 1). Since FM dye labelling of SVs is quantal¹⁰, the resulting fluorescence drop is proportional to the amount of vesicles released. Thus, the recycling and fusion of SVs generated from the previous round of endocytosis can be reliably quantified.Here, we present a protocol that has been modified to obtain two additional elements of information. Firstly, sequential unloading stimuli are used to differentially unload the RRP and the RP, to allow quantification of the replenishment of specific SV pools. Secondly, each nerve terminal undergoes the protocol twice. Thus, the response of the same nerve terminal at S1 can be compared against the presence of a test substance at phase S2 (Figure 2), providing an internal control. This is important, since the extent of SV recycling across different nerve terminals is highly variable¹¹.Any adherent primary neuronal cultures may be used for this protocol, however the plating density, solutions and stimulation conditions are optimised for cerebellar granule neurons (CGNs)^12,13.     

Complete parameter bounds and quasiidentifiability conditions for a class of unidentifiable linear systems

Lack of unique structural identifiability for parameters of dynamic system models is a very common situation with practical experimental schemes, particularly when studying biological systems. However, for well-structured (e.g., multicompartmental) models, it is often possible to localize unidentifiable parameters between finite limits (“interval identifiability”), using the same data base, and under certain conditions these limits nearly coincide. Two new results in this area are presented: (1) The smallest ranges on all unidentifiable rate constants and pool sizes of the most general n-compartment mammillary system are derived, in an easy-to-program algorithmic form, for the common case of input forcing and output measurements in the central pool only. From these results we see why elimination rate constants (“leaks”) are difficult to distinguish from zero, whereas exchange rate constants between pools, and pool sizes, may be bounded very tightly in certain circumstances. (2) The notion of quasiidentifiability, or sufficient identifiability for practical purposes, is introduced to quantify these circumstances. Each of the rate constants between central and peripheral pools, and all pool sizes, are quasiidentifiable if the magnitude of the ratio of the coefficient to the eigenvalue of the slowest mode is very much greater than the largest coefficient in the sum-of-exponentials response function. Also quasiidentifiability is a necessary condition for applicability of noncompartmental analysis to estimate pool sizes and residence times of mammillary systems with “leaky” noncentral pools.     

Bassoon‐disruption slows vesicle replenishment and induces homeostatic plasticity at a CNS synapse

Endbulb of Held terminals of auditory nerve fibers (ANF) transmit auditory information at hundreds per second to bushy cells (BCs) in the anteroventral cochlear nucleus (AVCN). Here, we studied the structure and function of endbulb synapses in mice that lack the presynaptic scaffold bassoon and exhibit reduced ANF input into the AVCN. Endbulb terminals and active zones were normal in number and vesicle complement. Postsynaptic densities, quantal size and vesicular release probability were increased while vesicle replenishment and the standing pool of readily releasable vesicles were reduced. These opposing effects canceled each other out for the first evoked EPSC, which showed unaltered amplitude. We propose that ANF activity deprivation drives homeostatic plasticity in the AVCN involving synaptic upscaling and increased intrinsic BC excitability. In vivo recordings from individual mutant BCs demonstrated a slightly improved response at sound onset compared to ANF, likely reflecting the combined effects of ANF convergence and homeostatic plasticity. Further, we conclude that bassoon promotes vesicular replenishment and, consequently, a large standing pool of readily releasable synaptic vesicles at the endbulb synapse.     

Dispensable,Redundant, Complementary,and Cooperative Roles of Dopamine,Octopamine, and Serotonin in Drosophila melanogaster

To investigate the regulation of Drosophila melanogaster behavior by biogenic amines, we have exploited the broad requirement of the vesicular monoamine transporter (VMAT) for the vesicular storage and exocytotic release of all monoamine neurotransmitters. We used the Drosophila VMAT (dVMAT) null mutant to globally ablate exocytotic amine release and then restored DVMAT activity in either individual or multiple aminergic systems, using transgenic rescue techniques. We find that larval survival, larval locomotion, and female fertility rely predominantly on octopaminergic circuits with little apparent input from the vesicular release of serotonin or dopamine. In contrast, male courtship and fertility can be rescued by expressing DVMAT in octopaminergic or dopaminergic neurons, suggesting potentially redundant circuits. Rescue of major aspects of adult locomotion and startle behavior required octopamine, but a complementary role was observed for serotonin. Interestingly, adult circadian behavior could not be rescued by expression of DVMAT in a single subtype of aminergic neurons, but required at least two systems, suggesting the possibility of unexpected cooperative interactions. Further experiments using this model will help determine how multiple aminergic systems may contribute to the regulation of other behaviors. Our data also highlight potential differences between behaviors regulated by standard exocytotic release and those regulated by other mechanisms.     

Minimal models of multidimensional computations

The multidimensional computations performed by many biological systems are often characterized with limited information about the correlations between inputs and outputs. Given this limitation, our approach is to construct the maximum noise entropy response function of the system, leading to a closed-form and minimally biased model consistent with a given set of constraints on the input/output moments; the result is equivalent to conditional random field models from machine learning. For systems with binary outputs, such as neurons encoding sensory stimuli, the maximum noise entropy models are logistic functions whose arguments depend on the constraints. A constraint on the average output turns the binary maximum noise entropy models into minimum mutual information models, allowing for the calculation of the information content of the constraints and an information theoretic characterization of the system's computations. We use this approach to analyze the nonlinear input/output functions in macaque retina and thalamus; although these systems have been previously shown to be responsive to two input dimensions, the functional form of the response function in this reduced space had not been unambiguously identified. A second order model based on the logistic function is found to be both necessary and sufficient to accurately describe the neural responses to naturalistic stimuli, accounting for an average of 93% of the mutual information with a small number of parameters. Thus, despite the fact that the stimulus is highly non-Gaussian, the vast majority of the information in the neural responses is related to first and second order correlations. Our results suggest a principled and unbiased way to model multidimensional computations and determine the statistics of the inputs that are being encoded in the outputs.     

Mobilization of the Readily Releasable Pool of Acetylcholine from a Sympathetic Ganglion by Tityustoxin in the Presence of Vesamicol

The present experiments tested whether preganglionic stimulation and direct depolarization of nerve terminals by tityustoxin could mobilize similar or different pools of acetylcholine (ACh) from the cat superior cervical ganglia in the presence of 2-(4-phenylpiperidino)cyclohexanol (vesamicol, AH5183), an inhibitor of ACh uptake into synaptic vesicles. In the absence of vesamicol, both nerve stimulation and tityustoxin increased ACh release. In the presence of vesamicol, the release of ACh induced by tityustoxin was inhibited, and just 16% of the initial tissue content could be released, a result similar to that obtained with electrical stimulation under the same condition. When the impulse-releasable pool of ACh had been depleted, tityustoxin still could release transmitter, amounting to some 10% of the ganglion's initial content. This pool of transmitter seemed to be preformed in the synaptic vesicles, rather than synthesized in response to stimuli, as tityustoxin could not release newly synthesized [3H]ACh formed in the presence of vesamicol, and hemicholinium-3 did not prevent the toxin-induced release. In contrast to the results with tityustoxin, preganglionic stimulation could not release transmitter when impulse-releasable or toxin-releasable compartments had been depleted. Our results confirm that vesamicol inhibits the mobilization of transmitter from a reserve to a more readily releasable pool, and they also suggest that, under these experimental conditions, there might be some futile transmitter mobilization, apparently to sites other than nerve terminal active zones.     

Analysis of Ca2+ fluxes and Ca2+ pools in pancreatic acini

45Ca2+ movements have been analysed in dispersed acini prepared from rat pancreas in a quasi-steady state for 45Ca2+. Carbamyl choline (carbachol; Cch) caused a quick 45Ca2+ release that was followed by a slower 45Ca2+ 'reuptake'. Subsequent addition of atropine resulted in a further transient increase in cellular 45Ca2+. The data suggest the presence of a Cch-sensitive 'trigger' pool, which could be refilled by the antagonist, and one or more intracellular 'storage' pools. Intracellular Ca2+ sequestration was studied in isolated acini pretreated with saponin to disrupt their plasma membranes. In the presence of 45Ca2+ (1 microM), addition of ATP at 5 mM caused a rapid increase in 45Ca2+ uptake exceeding the control by fivefold. Maximal ATP-promoted Ca2+ uptake was obtained at 10 microM Ca2+ (half-maximal at 0.32 microM Ca2+). In the presence of mitochondrial inhibitors it was 0.1 microM (half-maximal at 0.014 microM). 45Ca2+ release could still be induced by Cch but the subsequent reuptake was missing. The latter was restored by ATP and atropine caused further 45Ca2+ uptake. Electron microscopy showed electron-dense precipitates in the rough endoplasmic reticulum of saponin-treated cells in the presence of Ca2+, oxalate and ATP which were absent in intact cells or cells pretreated with A23187. The data suggest the presence of a plasma membrane-bound Cch-sensitive 'trigger' Ca2+ pool and ATP-dependent Ca2+ storage systems in mitochondria and rough endoplasmic reticulum of pancreatic acini. It is assumed that Ca2+ is taken up into these pools after secretagogue-induced Ca2+ release.U     

Reversible disruption of thymic function by steroid treatment

The effect of steroid treatment on the thymic output of T cells was examined in an avian model. Recent thymic emigrants in chickens transiently express the chicken T cell Ag 1 thymocyte marker, and thymic function can be monitored indirectly by measuring the levels of TCR gene rearrangement excision circles in peripheral T cells. Both parameters were used to show that intensive steroid treatment induces thymic involution and a profound reduction in the supply of naive T cells to the periphery. Conversely, resident T cells in the peripheral lymphocyte pool were relatively spared. Thymopoiesis immediately recovered following cessation of steroid treatment, concurrent with restoration of the thymic output of newly formed T cells. Repopulation of the peripheral T cell pool recapitulated the ontogenetic pattern of gamma delta T cell replenishment before alpha beta T cell reseeding, thereby indicating the complete recovery of thymic function after a course of steroid treatment.     

VESICULAR STORAGE AND RELEASE OF ACETYLCHOLINE IN TORPEDO ELECTROPLAQUE SYNAPSES

Abstract— The disposition of newly synthesized ACh subsequent to depletion of vesicular endogenous ACh by stimulation was studied in the electromotor nerve terminals of Torpedo marmorata using [³H]acetate as a precursor of ACh. Little vesicular [³H]ACh could be isolated from tissue immediately after stimulation at 1 Hz. After 3 h post-stimulation recovery the newly synthesized [³H]ACh is found predominantly in a subpopulation of vesicles distinct from the vesicles containing most of the endogenous poorly labelled ACh. Restimulation of the tissue causes release of highly labelled ACh with a specific radioactivity (SRA) comparable to that of the newly synthesized [³H]ACh in the highly labelled subpopulation of vesicles and significantly greater than the SRA of ACh in the main vesicular pool or the total tissue.     

GENETIC SCREENING FOR IDENTIFICATION OF SALMONELLA TYPHIMURIUM TN5 MUTANTS WITH POTENTIAL HYPERSENSITIVITY TO SHORT-CHAIN FATTY ACIDS

This study was conducted to test if transposon footprinting could be used to identify transposon mutants of Salmonella typhimurium with growth defects in a media containing short-chain fatty acids (SCFA) as the test selective condition. High concentrations of SCFA are one of the characteristic conditions in the animal intestine that has been suggested to play a role in inhibiting colonization by nonindigenous bacterial pathogens. When the mutant pools containing 25 Tn5 mutants/pool were analyzed for transposon footprints before and after selection, a polymerase chain reaction (PCR) product could be identified that was present in an input pool, but not in a corresponding output pool. The results indicate that transposon footprinting can be used for negative screening of genes sensitive to SCFA in the S. typhimurium bacterial genome.     

Nutrient dynamics and ecosystem metabolism in the Bay of Blanes (NW Mediterranean)

The dynamics of the nutrient pools and their stoichiometry as well as their control by ecosystem metabolism (benthic and planktonic) and benthic–pelagic exchanges (sedimentation rates and sediment waterfluxes) were examined in the Mediterranean littoral (Blanes Bay, NE Spain). Dissolved organic nitrogen comprised about half of the nitrogen present in the water column and the carbon pool was dominated by the inorganic pool (95% of the carbon present in the water column). The dissolved and particulate organic pools were deficient in P relative to C and N, indicating a rapid recycling of P from organic matter. The pelagic compartment was heterotrophic, supported by significant allochthonous inputs of land material, which also contributed greatly to the sedimentary inputs (37% of total sedimenting carbon). In contrast, the benthic compartment was autotrophic, with the excess net benthic community production balancing the deficit in pelagic community production, leading to metabolic equilibrium at the station studied. Sedimentary inputs of nitrogen, phosphorus and silicon exceeded the benthic release, indicating that the benthic compartment acted as a sink for nutrients, consistent with its autotrophic nature. Carbon inputs to the benthic compartment also exceeded requirements, due to the allochthonous subsidies to the system, so that the benthic compartment stored or exported organic carbon. An erratum to this article can be found at .     

Base cation biogeochemistry and weathering under oak and pine: a controlled long-term experiment

Large earthen-walled lysimeters at the San Dimas Experimental Forest in southern California present a unique opportunity to assess vegetation effects on biogeochemical processes and cation release by weathering in controlled soil-vegetation systems where archived samples of soil parent material are available for comparison. The lysimeters were filled in 1937 with homogenized fine sandy loam derived on site from the weathering of diorite, and planted in 1946 with scrub oak (Quercus dumosa) and Coulter pine (Pinus coulteri). Changes in base cation contents were measured in above-ground biomass, and total and exchangeable soil pools to a depth of 1 meter. All cations in the non-exchangeable soil pool decreased relative to the initial fill material, indicating release by weathering. Sodium and K were depleted from both exchangeable and non-exchangeable pools of the soils. Plant uptake of Na was minimal, whereas K storage in vegetation exceeded the loss from the exchangeable soil pool. In both soil-vegetation systems, but especially for oak, there was an increase in exchangeable Ca and Mg. For all base cations, storage in above-ground biomass was greater for oak, whereas losses by weathering from the non-exchangeable soil pool were greater under pine. Strong evidence supports biocycling as a controlling mechanism resulting in greater Ca and Mg release by weathering under pine. In addition, decreases in non-exchangeable Ca and Mg were strongly correlated to decrease in Si under oak, whereas no correlation was observed under pine. We conclude that weathering reactions or stoichiometry differed between vegetation types.Corresponding author     

Acetylcholine compartments in mouse diaphragm. Comparison of the effects of black widow spider venom, electrical stimulation, and high concentrations of potassium

We have studied the effects of 25 mM potassium, electrical stimulation of the phrenic nerve, and crude black widow spider venom on the ultrastructure, electrophysiology, and acetylcholine (ACh) contents of mouse diaphragms. About 65% of the ACh in diaphragms is contained in a depletable store in the nerve terminals. The rest of the ACh is contained in a nondepletable store that may correspond to the store that remains in denervated muscles and includes, in addition, ACh in the intramuscular branches of the phrenic nerve. About 4% of the ACh released from the depletable store at rest is secreted as quanta and may come from the vesicles, while 96% is secreted in a nonquantized form and comes from an extravesicular pool. The size of the extravesicular pool is uncertain: it could be less than 10%, or as great as 50%, of the depletable store. K causes a highly (but perhaps not perfectly) selective increase in the rate of quantal secretion so that quanta account for about 50% of the total ACh released from K- treated diaphragms. K, or electrical stimulation of the phrenic nerve, depletes both the vesicular and extravesicular pools of ACh when hemicholinium no. 3 (HC-3) is present. However, most of the vesicles are retained under these conditions so that the diaphragms are able to increase slightly their rates of release of ACh when K is added. Venom depletes the terminals of their vesicles and abolishes the release of quanta of ACh. It depletes the vesicular pool of ACh (since it depletes the vesicles), but may only partially deplete the extravesicular pool (since it reduces resting release only 10--40%). The rate of release of ACh from the residual extravesicular pool does not increase when 25 mM K is added. Although we cannot exclude the possibility that stimulation may double the rate of release of ACh from the extravesicular pool, our results are compatible with the idea that the ACh released by stimulation comes mainly from the vesicles and that, when synthesis is inhibited by HC-3, ACh may be exchanged between the extravesicular pool and recycled vesicles.     

Fluxes of terrestrial and aquatic carbon by emergent mosquitoes: a test of controls and implications for cross-ecosystem linkages

Adult aquatic insects are a common resource for many terrestrial predators, often considered to subsidize terrestrial food webs. However, larval aquatic insects themselves consume both aquatic primary producers and allochthonous terrestrial detritus, suggesting that adults could provide aquatic subsidy and/or recycled terrestrial energy to terrestrial consumers. Understanding the source of carbon (aquatic vs. terrestrial) driving aquatic insect emergence is important for predicting magnitude of emergence and effects on recipient food web dynamics; yet direct experimental tests of factors determining source are lacking. Here, we use Culex mosquitoes in experimental pools as an exemplar to test how variation in general factors common to aquatic systems (terrestrial plant inputs and light) may alter the source and amount of energy exported to terrestrial ecosystems in adult aquatic insects that rely on terrestrial resources as larvae. We found strong sequential effects of terrestrial plant inputs and light on aquatic insect oviposition, diet, and emergence of Culex mosquitoes. Ovipositing mosquitoes laid ~3 times more egg masses in high terrestrial input pools under low light conditions. This behavior increased adult emergence from pools under low light conditions; however, high input pools (which had the highest mosquito densities) showed low emergence rates due to density-dependent mortality. Mosquito diets consisted mainly of terrestrial resources (~70–90 %). As a result, the amount of aquatic carbon exported from pools by mosquitoes during the experiment was ~18 times higher from low versus high light pools, while exports of terrestrial carbon peaked from pools receiving intermediate levels of inputs (3–6 times higher) and low light (~6 times higher). Our results suggest that understanding the interplay among terrestrial plant inputs, light availability and biotic responses of aquatic insects may be key in predicting source and magnitude of emergence, and thus the strength and effects of aquatic–terrestrial linkages in freshwater systems.     

Effects of Reserpine and Tetrabenazine on Catecholamine and ATP Storage in Cultured Bovine Adrenal Medullary Chromaffin Cells

The in vivo storage relationship between catecholamines and ATP in chromaffin vesicles of cultured bovine adrenal medulla cells was investigated using drugs that block vesicular catecholamine uptake. Three-day treatments with reserpine and tetrabenazine causing 85-90% depletion of catecholamines resulted in 41-46% reductions in cellular ATP content. Subcellular fractionation of reserpine-treated cells indicated that the ATP is lost from the chromaffin vesicle pool. This was confirmed in experiments using metabolic inhibitors to differentiate the vesicular and extravesicular ATP pools. The vesicular ATP loss was not proportional to that of catecholamines, resulting in a reduction by 50% in the chromaffin vesicle mole ratio of catecholamines to ATP after 48 h of treatment. In metabolic labeling studies, it was found that reserpine treatment reduced the incorporation of [3H]adenosine into vesicular ATP selectively, but it reduced the incorporation of 32Pi into both the vesicular and extravesicular pools. The reduction of the [3H]adenosine incorporation was not due to diminished vesicular nucleotide uptake resulting from low catecholamine levels, because when the catecholamines were depleted by tetrabenazine pretreatment followed by removal of the drug before labeling, no reduction in [3H]adenosine incorporation was observed. When present during the labeling, tetrabenazine was found to be a reversible inhibitor of plasma membrane adenosine uptake. The observed loss of adenine nucleotides from catecholamine-depleted chromaffin vesicles in vivo provides evidence that interactions between ATP and catecholamines are important in the vesicular storage of high concentration of these compounds.