Effect of nitrogen supply on stay-green sorghum in differing post-flowering water regimes.

MAIN CONCLUSION: The expression of stay-green (SG) characteristic in sorghum under water stress was related to N supply. SG genotype performed better than a non-stay-green (NSG) genotype at medium and high N levels. The differences in physiological parameters between SG and NSG genotypes were not significant at low N level and severe water stress. Grain sorghum [Sorghum bicolor (L.) Moench] with stay-green (SG) trait has the potential to produce more biomass and use soil water and nitrogen (N) more efficiently under post-flowering water stress. Previous studies were mostly conducted without N deficiency and more information is needed for interactions among soil N availability, SG genotype, and post-flowering water stress. In this study, the differences in leaf growth and senescence, shoot and root biomass, evapotranspiration (ET), water use efficiency (WUE), leaf photosynthetic responses, and nitrogen use efficiency (NUE) between a SG genotype (BTx642) and a non-stay-green (NSG) genotype (Tx7000) were examined. The two genotypes were grown at three N levels (Low, LN; Medium, MN; High, HN) and under three post-flowering water regimes (No water deficit, ND; Moderate water deficit, MD; Severe water deficit, SD). The genotypic difference was generally significant while it frequently interacted with N levels and water regimes. At medium and high N levels, SG genotype consistently had greater green leaf area, slower senescence rate, more shoot biomass and root biomass, and greater WUE and NUE than the NSG genotype under post-flowering drought. However, differences in several variables (e.g., leaf senescence, ET, WUE and NUE) between genotypes were not significant under SD at LN. At HN and MN, photosynthetic function of SG genotype was better maintained under drought. At LN, SG genotype maintained greater green leaf area but had lower photosynthetic activity than the NSG genotype. Nonetheless, adequate N supply is important for SG genotype under drought and greater root biomass may contribute to greater NUE in SG genotype.

Infection by Nanophyetus salmincola and Toxic Contaminant Exposure in Out-migrating Steelhead from Puget Sound, Washington: Implications for Early Marine Survival.

Out-migrating steelhead Oncorhynchus mykiss from four Puget Sound rivers and associated marine basins of Puget Sound in Washington State were examined for the parasite, Nanophyetus salmincola in 2014 to determine whether recent trends in reduced marine survival are associated with the presence of this pathogen. A subset of steelhead from three of these river-marine basin combinations was analyzed for the presence of persistent organic pollutants (POPs) to assess whether exposure to these contaminants is a contributing factor to their reduced marine survival. The prevalence and parasite load of N. salmincola were significantly higher in fish from central and southern Puget Sound than in fish from river systems in northern Puget Sound. The proportion of steelhead samples with concentrations of POPs higher than adverse effects thresholds (AETs) or concentrations known to cause adverse effects was also greater in fish from the central and southern regions of Puget Sound than in those from the northern region. Polybrominated diphenyl ether concentrations associated with increased disease susceptibility were observed in 10% and 40% of the steelhead sampled from central and southern Puget Sound regions, respectively, but in none of the fish sampled from the northern region. The AET for polychlorinated biphenyls was exceeded in steelhead collected from marine habitats: 25% of the samples from the marine basins in the central and southern regions of Puget Sound and 17% of samples from northern Puget Sound region. Both N. salmincola and POP levels suggest there are adverse health effects on out-migrating steelhead from one southern and one central Puget Sound river that have lower early marine survival than those from a river system in northern Puget Sound.

A multiple data set phylogeny for the endemic South African freshwater phreatoicidean isopod genus Mesamphisopus: Taxonomic and biogeographic implications.

The obligate, freshwater isopod suborder Phreatoicidea is represented in South Africa by ten species contained within the endemic genus Mesamphisopus (Mesamphisopidae). Here, phylogenetic hypotheses are proposed to describe the evolutionary and biogeographic history of the genus with respect to drainage basin evolution and to assess species diversity, particularly among populations variably identified as Mesamphisopusabbreviatus or Mesamphisopusdepressus. Twenty-three ingroup taxa were examined, including eight known species and representatives of the M. abbreviatus-depressus complex. Allozyme data from 12 loci were analysed phenetically and cladistically. Mitochondrial DNA sequence data from the 12S ribosomal RNA and cytochrome c oxidase subunit I genes were analysed as a combined mtDNA data set and as a total data set in combination with recoded allele frequency data. Analyses retrieved (1) a monophyletic Mesamphisopus; (2) Mesamphisopustsitsikamma and a Mesamphisopuspaludosus+Mesamphisopuspenicillatus clade as basal lineages; (3) a Mesamphisopuscapensis+Mesamphisopusbaccatus clade; and (4) a clade containing the M. abbreviatus-depressus complex, with these taxa nested among several other species. Large genetic distances among taxa and the paraphyly of the members of the M. abbreviatus-depressus complex suggested the presence of hidden taxonomic diversity in Mesamphisopus. Clear biogeographic patterns emerged with lineages and clades mostly restricted to geographically discrete regions. Patterns showed remarkable similarity to those seen in the region's terrestrial fauna and bore no relation to the history of drainage basins. These patterns suggested that vicariance and, possibly, limited dispersal events played a major role in the evolution of Mesamphisopus.

Physiological mechanisms contributing to the increased water-use efficiency in winter wheat under deficit irrigation.

Deficit irrigation in winter wheat has been practiced in the areas with limited irrigation water resources. The objectives of this study were to (i) understand the physiological basis for determinations of grain yield and water-use efficiency in grain yield (WUE) under deficit irrigation; and (ii) investigate the effect of deficit irrigation on dry matter accumulation and remobilization of pre-anthesis carbon reserves during grain filling. A field experiment was conducted in the Southern High Plains of the USA and winter wheat (cv. TAM 202) was grown on Pullman clay loam soil (fine mixed thermic Torretic Paleustoll). Treatments consisted of rain-fed, deficit irrigation from jointing to the middle of grain filling, and full irrigation. The physiological measurements included leaf water potential, net photosynthetic rate (Pn), stomatal conductance (Gs), and leaf area index. The rain-fed treatment had the lowest seasonal evapotranspiration (ET), biomass, grain yield, harvest index (HI) and WUE as a result of moderate to severe water stress from jointing to grain filling. Irrigation application increased seasonal ET, and ET increased as irrigation frequency increased. The seasonal ET increased 20% in one-irrigation treatments between jointing and anthesis, 32-46% in two-irrigation treatments, and 67% in three- and full irrigation treatments. Plant biomass, grain yield, HI and WUE increased as the result of increased ET. The increased yield under irrigation was mainly contributed by the increased number of spikes, and seeds per square meter and per spike. Among the irrigation treatments, grain yield increased significantly but the WUE increased slightly as irrigation frequency increased. The increased WUE under deficit irrigation was contributed by increased HI. Water stress during grain filling reduced Pn and Gs, and accelerated leaf senescence. However, the water stress during grain filling induced remobilization of pre-anthesis carbon reserves to grains, and the remobilization of pre-anthesis carbon reserves significantly contributed to the increased grain yield and HI. The results of this study showed that deficit irrigation between jointing and anthesis significantly increased wheat yield and WUE through increasing both current photosynthesis and the remobilization of pre-anthesis carbon reserves.

Effect of estrogen deficiency on skeletal and alveolar bone density in sheep.

BACKGROUND: This study provides a longitudinal assessment of changes in alveolar and skeletal bone mineral density (BMD) in ovariectomized animals. METHODS: Following ovariectomy (OVX) (n = 6) or sham-operation (n = 6) intraoral radiographs were made at 4-month intervals and serum 17-beta-estradiol, osteocalcin, and interleukin (IL)-6, urinary deoxypyridinium, and salivary IL-6, deoxypyridinium, and osteocalcin concentrations were evaluated. Twelve months after surgery, animals were sacrificed and the mandible and radius/ulna removed. Bones were sectioned and radiographed. Mean BMD and cortical thicknesses were calculated from each region. RESULTS: OVX animals had a progressive decrease in serum 17-beta-estradiol, increased serum osteocalcin and IL-6, urinary deoxypyridinium and salivary IL-6, osteocalcin and deoxypyridinium (P < 0.001), suggesting that they were becoming osteoporotic. The BMD of the radius/ulna and mandibular alveolar bone was significantly reduced in OVX animals (P < 0.05 and P < 0.001, respectively). Reduced alveolar bone BMD became evident in OVX animals 6 months after surgery and became more severe during the subsequent 6 months. Alveolar crestal height was also significantly reduced in OVX animals (P < 0.001). These biochemical and density changes preceded a significant reduction in serum 17-beta-estradiol, which occurred between 4 and 8 months following surgery. CONCLUSIONS: Serial measurements of alveolar BMD predicts loss of skeletal BMD in OVX sheep. Changes in alveolar BMD precede estrogen deficiency, suggesting that early signs of reduced BMD may be detected in peri-menopausal women. The presence of biomarkers of bone metabolism within saliva and their correlation with reduced BMD suggests that saliva could be used as an adjunct screening method for assessment of skeletal bone density.

Drosophila Amphiphysin is a post-synaptic protein required for normal locomotion but not endocytosis.

Clathrin-mediated endocytosis is required to recycle synaptic vesicles for fast and efficient neurotransmission. Amphiphysins are thought to be multiprotein adaptors that may contribute to this process by bringing together many of the proteins required for endocytosis. Their in vivo function, however, has yet to be determined. Here, we show that the Drosophila genome encodes a single amphiphysin gene that is broadly expressed during development. We also show that, unlike its vertebrate counterparts, Drosophila Amphiphysin is enriched postsynaptically at the larval neuromuscular junction. To determine the role of Drosophila Amphiphysin, we also generated null mutants which are viable but give rise to larvae and adults with pronounced locomotory defects. Surprisingly, the locomotory defects cannot be accounted for by alterations in the morphology or physiology of the neuromuscular junction. Moreover, using stimulus protocols designed to test endocytosis under moderate and extreme vesicle cycling, we could not detect any defect in the neuromuscular junction of the amphiphysin mutant. Taken together, our findings suggest that Amphiphysin is not required for viability, nor is it absolutely required for clathrin-mediated endocytosis. However, Drosophila Amphiphysin function is required in both larvae and adults for normal locomotion.

Two distinct effects on neurotransmission in a temperature-sensitive SNAP-25 mutant.

Vesicle fusion in eukaryotic cells is mediated by SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors). In neurons, the t-SNARE SNAP-25 is essential for synaptic vesicle fusion but its exact role in this process is unknown. We have isolated a SNAP-25 temperature-sensitive paralytic mutant in Drosophila, SNAP-25(ts). The mutation causes a Gly50 to Glu change in SNAP-25's first amphipathic helix. A similar mutation in the yeast homologue SEC9 also results in temperature sensitivity, implying a conserved role for this domain in secretion. In vitro-generated 70 kDa SNARE complexes containing SNAP-25(ts) are thermally stable but the mutant SNARE multimers (of approximately 120 kDa) rapidly dissociate at 37 degrees C. The SNAP-25(ts) mutant has two effects on neurotransmitter release depending upon temperature. At 22 degrees C, evoked release of neurotransmitter in SNAP-25(ts) larvae is greatly increased, and at 37 degrees C, the release of neurotransmitter is reduced as compared with controls. Our data suggest that at 22 degrees C the mutation causes the SNARE complex to be more fusion competent but, at 37 degrees C the same mutation leads to SNARE multimer instability and fusion incompetence.

Analysis of the mutant Drosophila N-ethylmaleimide sensitive fusion-1 protein in comatose reveals molecular correlates of the behavioural paralysis.

NEM-sensitive fusion protein (NSF) is an ATPase required for many intracellular membrane trafficking steps. Recent studies have suggested that NSF alters the conformation of the SNAP receptors (SNAREs) to permit their interaction, or to uncouple them after they interact. Most organisms have a single NSF gene product but Drosophila express two highly related isoforms, dNSF-1 and dNSF-2. dNSF-1 is encoded by the gene comatose (comt), first identified as the locus of a temperature-sensitive paralytic mutation. Here we show that dNSF-1 is most abundant in the nervous system and can be detected in larval and adult CNS. Subcellular fractionation revealed that dNSF-1 was enriched in a vesicle fraction along with the synaptic vesicle protein synaptotagmin. comt flies maintained at the non-permissive temperature rapidly accumulate sodium dodecyl sulfate (SDS)-resistant SNARE complexes at the restrictive temperature, with concomitant translocation of dNSF-1 from cytosol and membrane fractions into a Triton X-100 insoluble fraction. The long recovery of comt flies after heat shock induced paralysis correlated with the irreversibility of this translocation. Interestingly, while dNSF-1 also translocates in comt(TP7) larvae, there is no associated neurophysiological phenotype at the neuromuscular junction (nmj) or accumulation of SDS-resistant complexes in the CNS. Together, these results suggest that dNSF-1 is required for adult neuronal function, but that in the larval nmj function may be maintained by other isoforms.

SNARE-dependent signaling at the Drosophila wing margin.

The wing of Drosophila melanogaster has long been used as a model system to characterize intermolecular interactions important in development. Implicit in our understanding of developmental processes is the proper trafficking and sorting of signaling molecules, although the precise mechanisms that regulate membrane trafficking in a developmental context are not well studied. We have therefore chosen the Drosophila wing to assess the importance of SNARE-dependent membrane trafficking during development. N-Ethylmaleimide-sensitive fusion protein (NSF) is a key component of the membrane-trafficking machinery and we constructed a mutant form of NSF whose expression we directed to the developing wing margin. This resulted in a notched-wing phenotype, the severity of which was enhanced when combined with mutants of VAMP/Synaptobrevin or Syntaxin, indicating that it results from impaired membrane trafficking. Importantly, we find that the phenotype is also enhanced by mutations in genes for wingless and components of the Notch signaling pathway, suggesting that these signaling pathways were disrupted. Finally, we used this phenotype to conduct a screen for interacting genes, uncovering two Notch pathway components that had not previously been linked to wing development. We conclude that SNARE-mediated membrane trafficking is an important component of wing margin development and that dosage-sensitive developmental pathways will act as a sensitive reporter of partial membrane-trafficking disruption.

SNARE proteins contribute to calcium cooperativity of synaptic transmission.

A hallmark of calcium-triggered synaptic transmission is the cooperative relationship between calcium and the amount of transmitter released. This relationship is thought to be important for improving the efficiency of synaptic vesicle exocytosis. Although it is generally held that cooperativity arises from the interaction of multiple calcium ions with a single calcium-sensing molecule, the precise molecular basis of this phenomenon is not known. The SNARE proteins are known to be critical for synaptic vesicle exocytosis. We therefore tested for a contribution of SNARE proteins to cooperativity by genetically reducing the levels of syntaxin IA and neuronal-synaptobrevin in Drosophila. Surprisingly, we found that reducing these SNARE proteins also reduced Ca(2+) cooperativity. Thus, SNARE proteins are important for determining the cooperative relationship between calcium and synaptic transmission.

Demonstration of in vivo bioequivalence of a generic albuterol metered-dose inhaler to Ventolin.

STUDY OBJECTIVE: To use histamine bronchoprovocation and bioassay statistical procedures to evaluate the in vivo bioequivalence of a generic albuterol metered-dose inhaler (MDI). DESIGN: A randomized, double-blind, balanced, crossover design was used to determine the potency of each generic albuterol MDI actuation relative to Ventolin (Glaxo Wellcome; Research Triangle Park, NC) administration. One treatment was administered on each of 4 study days. A histamine bronchoprovocation procedure was initiated 1.25 h before and 15 min after administration of the study treatment. PATIENTS: Twenty-four nonsmoking subjects with mild-to-moderate asthma were studied (18 to 65 years of age; FEV(1), > 60% of predicted; and provocative concentration of histamine causing a 20% fall in FEV(1) [PC(20)], < or = 8 mg/mL at screening). INTERVENTIONS: One and four actuations (90 and 360 microg, respectively) of the generic MDI and of Ventolin MDI. Placebo inhalers were used to maintain blinding of inhaler and doses. MEASUREMENTS AND RESULTS: The primary outcome variable was histamine PC(20) measured after study treatment administration. A significant dose-effect relationship was present (p < 0.0001). Deviation from parallelism of the generic and Ventolin dose-response curves (p = 0.95) and differences in overall mean response between the two formulations (p = 0.68) were not significant. Using Finney 2 x 2 bioassay statistical procedures, we estimated that one actuation of the generic albuterol MDI was equivalent to 1.01 puffs of Ventolin (90% confidence interval, 0.69 to 1.50). CONCLUSION: The generic albuterol MDI delivers a quantity of albuterol to the beta(2)-receptor site in the lung that is the bioequivalent to Ventolin. Further, this study reinforces the validity of this statistical methodology for determining in vivo bioequivalence.

Relationship between mutations in parC and gyrA of clinical isolates of Streptococcus pneumoniae and resistance to ciprofloxacin and grepafloxacin.

The mechanisms of resistance to ciprofloxacin and grepafloxacin were studied in 54 clinical isolates of Streptococcus pneumoniae. Restriction fragment length polymorphism analysis following HinfI digestion was used with DNA sequencing to identify mutations in the quinolone resistance-determining regions (QRDRs) of the parC and gyrA genes. Ciprofloxacin MICs up to 16 mg/L were not associated with mutations to these genes in approximately half of the isolates. In other isolates, moderate levels of ciprofloxacin resistance (MIC 8-16mg/L) were associated with an alteration of ParC, most commonly entailing replacement of serine-79 by phenylalanine. High-level ciprofloxacin resistance (MIC 32-128 mg/L) entailed the additional mutation of GyrA with substitution of serine-83 by phenylalanine. Grepafloxacin MICs >4 mg/L were associated with this GyrA mutation alone; no relationship was detected between grepafloxacin MICs and mutation of the QRDR of parC.

Distinct requirements for evoked and spontaneous release of neurotransmitter are revealed by mutations in the Drosophila gene neuronal-synaptobrevin.

Two modes of vesicular release of transmitter occur at a synapse: spontaneous release in the absence of a stimulus and evoked release that is triggered by Ca2+ influx. These modes often have been presumed to represent the same exocytotic apparatus functioning at different rates in different Ca2+ concentrations. To investigate the mechanism of transmitter release, we have examined the role of synaptobrevin/VAMP, a protein involved in vesicular docking and/or fusion. We generated a series of mutations, including null mutations, in neuronal-synaptobrevin (n-syb), the neuronally expressed synaptobrevin gene in Drosophila. Mutant embryos completely lacking n-syb form morphologically normal neuromuscular junctions. Electrophysiological recordings from the neuromuscular junction of these mutants reveal that the excitatory synaptic current evoked by stimulation of the motor neuron is abolished entirely. However, spontaneous release of quanta from these terminals persists, although its rate is reduced by 75%. Thus, at least a portion of the spontaneous "minis" that are seen at the synapse can be generated by a protein complex that is distinct from that required for an evoked synaptic response.

Regulated spacing of synapses and presynaptic active zones at larval neuromuscular junctions in different genotypes of the flies Drosophila and Sarcophaga.

Synapses at larval neuromuscular junctions of the flies Drosophila melanogaster and Sarcophaga bullata are not distributed randomly. They have been studied in serial electron micrographs of two identified axons (axons 1 and 2) that innervate ventral longitudinal muscles 6 and 7 of the larval body wall. The following fly larvae were examined: axon 1--wild-type Sarcophaga and Drosophila and Drosophila mutants dunce(m14) and fasII(e76), a hypomorphic allele of the fasciclin II gene; and axon 2--drosophila wild-type, dunce(m14), and fasII(e76). These lines were selected to provide a wide range of nerve terminal phenotypes in which to study the distribution and spacing of synapses. Each terminal varicosity is applied closely to the underlying subsynaptic reticulum of the muscle fiber and has 15-40 synapses. Each synapse usually bears one or more active zones, characterized by dense bodies that are T-shaped in cross section; they are located at the presumed sites of transmitter release. The distribution of synapses was characterized from the center-to-center distance of each synapse to its nearest neighbor. The mean spacing between nearest-neighbor pairs ranged from 0.84 microm to 1.05 microm for axon 1, showing no significant difference regardless of genotype. The corresponding values for axon 2, 0.58 microm to 0.75 microm, were also statistically indistinguishable from one another in terminals of different genotype but differed significantly from the values for axon 1. Thus, the functional class of the axon provides a clear prediction of the spacing of its synapses, suggesting that spacing may be determined by the functional properties of transmission at the two types of terminals. Individual dense bodies were situated mostly at least 0.4 microm away from one another, suggesting that an interaction between neighboring active zones could prevent their final positions from being located more closely.

Homeostasis of synaptic transmission in Drosophila with genetically altered nerve terminal morphology.

We present a new test of the hypothesis that synaptic strength is directly related to nerve terminal morphology through analysis of synaptic transmission at Drosophila neuromuscular junctions with a genetically reduced number of nerve terminal varicosities. Synaptic transmission would decrease in target cells with fewer varicosities if there is a relationship between the number of varicosities and the strength of synaptic transmission. Animals that have an extreme hypomorphic allele of the gene for the cell adhesion molecule Fasciclin II possess fewer synapse-bearing nerve terminal varicosities; nevertheless, synaptic strength is maintained at a normal level for the muscle cell as a whole. Fewer failures of neurotransmitter release and larger excitatory junction potentials from individual varicosities, as well as more frequent spontaneous release and larger quantal units, provide evidence for enhancement of transmitter release from varicosities in the mutant. Ultrastructural analysis reveals that mutant nerve terminals have bigger synapses with more active zones per synapse, indicating that synaptic enlargement and an accompanying increase in synaptic complexity provide for more transmitter release at mutant varicosities. These results show that morphological parameters of transmitting nerve terminals can be adjusted to functionally compensate for genetic perturbations, thereby maintaining optimal synaptic transmission.

Quantal measurement and analysis methods compared for crayfish and Drosophila neuromuscular junctions, and rat hippocampus.

Quantal content of transmission was estimated for three synaptic systems (crayfish and Drosophila neuromuscular junctions, and rat dentate gyrus neurons) with three different methods of measurement: direct counts of released quanta, amplitude measurements of evoked and spontaneous events, and charge measurements of evoked and spontaneous events. At the crayfish neuromuscular junction, comparison of the three methods showed that estimates from charge measurements were closer to estimates from direct counts, since amplitude measurements were more seriously affected by variable latency in evoked release of quantal units. Thus, charge measurements are better for estimating quantal content when direct counts cannot be made, as in crayfish at high frequency of stimulation or in the dentate gyrus neurons. At the Drosophila neuromuscular junction, there is almost no latency variation of quantal release in realistic physiological solutions, and the methods based upon amplitudes and charge give similar results. Distributions of evoked synaptic quantal events obtained by direct counts at the crayfish neuromuscular junction were compared to statistical distributions obtained by best fits. Binomial distributions with uniform or non-uniform probabilities of release generally provided good fits to the observations. From best fit distributions, the quantal parameters n (number of release sites) and p (their probability of release) can be calculated. We used two algorithms to estimate n and p: one allows for non-uniform probability of release and uses a modified chi-square (chi 2) criterion, and the second assumes uniform probability of release and derives parameters from maximum likelihood estimation (MLE). The bootstrap estimate of standard errors is used to determine the accuracy of n and p estimates.

Differential physiology and morphology of motor axons to ventral longitudinal muscles in larval Drosophila.

Morphological and physiological characteristics of the two major motor axons supplying the commonly studied ventral longitudinal muscle fibers (6 and 7) of third-instar Drosophila melanogaster larvae were investigated. The innervating terminals of the two motor axons differ in the size of their synapse-bearing varicosities. The terminal with the larger varicosities also fluoresces more brightly when stained with the vital fluorescent dye 4-(4-diethylaminostyryl)-N-methylpyridinium iodide (4-Di-2-Asp) and occupies a larger total contact area on the muscle fiber. Through selective simultaneous recording of synaptic currents from identified boutons in living preparations during elicitation of synaptic potentials, it was shown that the axon with the smaller varicosities generates a large excitatory junction potential (EJP) in muscle 6 and that the axon with the larger varicosities generates a smaller EJP. Short-term facilitation is more pronounced for the smaller EJP. In preparations treated with 4-Di-2-Asp, the fluorescence of smaller varicosities increases with stimulation that elicits the large EJPs, indicating an activity-dependent entry of calcium that enhances mitochondrial fluorescence. The differences in morphology and physiology of the two axons are similar to, though less pronounced than, those observed in "phasic" and "tonic" motor axons of crustaceans.

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 Na:K 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.