The development of nerve-muscle junctions in Rana catesbeiana tadpoles

The physiological properties of developing nerve-muscle junctions in Rana catesbeiana tadpoles are described. Developing neurons at different stages of ontogeny formed functional synaptic connections with a section of tail muscle implanted in place of the hind limb bud. Transmission is quantal in nature, sensitive in normal ways to calcium and magnesium concentrations, and conforms to a Poisson distribution. The quantal content is initially low and increases with development. Mepp's occur randomly and have low frequencies which increase slightly with development. The size of a single quantum of transmitter does not change during development. The muscle fibers are multiply innervated, resulting in Epp's with distinct peaks and complex skewed mepp amplitude histograms. No significant increases were observed in the level of differentiation of the developing motor neurons as a result of their having innervated a portion of mature tail muscle. The numbers of developing motor neurons increased in the experimental lateral motor column, and a lag in their maturity was observed relative to motor neurons in the control lateral motor column.     

The Quantal Nature of Synaptic Transmission at the Neuromuscular Junction of a Spider

Spontaneous and evoked release of transmitter at neuromuscular junctions in three different leg muscles of a tarantula (Dugesiella hentzi) was investigated. In most cases the spontaneous miniature potentials were released independently, although bursts from single synaptic junctions occasionally occurred. In contrast to recent findings in other arthropod muscles, focal extracellular recording from junctional areas revealed that the evoked release of transmitter quanta followed Poisson's theorem at low quantal content synaptic junctions in arachnid muscles.     

Interactions between dissociated rat sympathetic neurons and skeletal muscle cells developing in cell culture: II. Synaptic mechanisms

Dissociated rat sympathetic neurons and skeletal myotubes were grown in mass cultures and microcultures as described in the accompanying paper (C. A. Nurse, 1981, Develop. Biol.88, 55–70). Excitatory synaptic interactions developed between neuron and neuron and between neuron and myotube. Electrical coupling occurred rarely. More often, the interaction was chemical and as shown in the accompanying paper, cholinergic. At the chemical neuronmyotube junctions, spontaneous miniature potentials (mejp's), sensitive to d-tubocurarine, occurred infrequently (1–20/min) and their discharge appeared random; their amplitude distribution was skew at all ages (up to ca. 4 weeks) even when the myotube was innervated by a single neuron in microculture. The evoked postsynaptic potentials (ejp's) in the myotubes were sensitive in conventional ways to the extracellular calcium (Ca_o) and magnesium (Mg_o) concentrations, and several tests suggested that transmission was quantal. In a few cases where a single neuron innervated a myotube in microculture, the estimated mean quantal unit size (assuming “Poisson” release) was similar to the mode of the spontaneous mejp amplitude histogram, suggesting that many of the spontaneous units were similar to the evoked units. At several junctions the quantal content m_o, estimated by the “failure” method, varied nonlinearly with Ca_o over the range 0.2–1.2 mM; data could be fitted by a power relation where the power ranged from 2.6 to 5.2.     

Reduction of linolenate content in soybean cotyledons by a substituted pyridazinone

The effect of a substituted pyridazinone, 4-chloro-5-(dimethylamino)-2-phenyl-3(2H)-pyridazinone (San 9785), on the reduction of linolenate content was examined in the cotyledons of the soybean cv. Century and a low linolenate mutant of soybeans, C1640, cultured in vitro. No apparent changes in dry matter and total fatty acid accumulation were observed in the cotyledons developing in the presence of San 9785. However, a significant reduction of linolenate content with a corresponding increase in linoleate resulted from growth of the cotyledons in culture medium containing San 9785. San 9785 had a greater effect on decreasing the linolenate content in cotyledons excised in early developmental phases than later stages from both the wild type and mutant soybeans. This result supports other observations that the biosynthesis oflinolenate relative to the other major fatty acids of soybean cotyledons declines in later developmental stages. The compound becomes progressively less effective in reducing the content of linolenate during seed development in the mutant than in the wild type. Both San 9785 and the mutation result in changes in phosphatidylcholine molecular species which indicates the presence of a number oflinolenate desaturation systems in developing soybean cotyledons. The possible biochemical nature of the mutation is discussed.     

The effects of ethanol on pre-synaptic components of synaptic transmission in a model glutamatergic synapse: the crayfish neuromuscular junction

We have elucidated some of the mechanisms by which ethanol (EtOH) reduces synaptic efficacy at model glutamatergic synapses. The crayfish phasic and tonic neuromuscular junctions are superb models for directly assessing the effects of EtOH on pre-synaptic components of synaptic transmission. The ability to perform quantal analysis of synaptic transmission has allowed us to assess pre-synaptic alterations of release. Using this system, we report that the application of EtOH, within a range observed in intoxicated humans (44 and 88 mM), resulted in a diminution of excitatory post-synaptic potentials (EPSP) amplitudes. Additionally, using focal macro-patch recordings, quantal synaptic currents were recorded to assess the pre-synaptic component as potential target sites for EtOH's action. At the tonic neuromuscular junctions, EtOH (88 mM) reduced the probability of release (p), and in some cases, reduced the number of the release sites (n), but did not alter facilitation index nor did it affect the latency of vesicular release. At the phasic neuromuscular junction, a reduction in synaptic charge occurred during the presence of EtOH. Thus, the observed decrease in synaptic strength is at least partially attributable to a pre-synaptic alteration, specifically the release of fewer vesicles.     

Attaining and maintaining strong vocal synapses in female Xenopus laevis

Synaptic efficacy at the laryngeal neuromuscular synapse differs markedly in adult male and female Xenopus laevis. Here, we examined the relation between circulating estrogen and synapse strength in developing and adult female frogs. Circulating estrogen levels in males and females during juvenile and adult stages were measured using radioimmunoassays. Synaptic strength was determined by quantal analysis in isolated female larynges. In males, estrogen levels are low (<40 pg/mL) throughout development. In females, estrogen levels are similar to those in males until 9 months after metamorphosis is complete and then increase throughout development. Female laryngeal synapses have low quantal contents until 24 months; quantal content increases significantly between 24 and 26 months, and high quantal contents are maintained thereafter. Measures of reproductive maturation, ovary, and oviduct weights, are strongly and positively correlated with estrogen level in 16- to 26-month females, while oocyte maturation is age dependent. Estrogen level and quantal content are not well correlated in these females. Ovariectomy at 24 months prevents the expected increase in quantal content and ovariectomy at 28 months results in a decrease in quantal content. Thus, the sex difference in efficacy of the laryngeal synapse develops under the influence of the ovary and requires the ovary for maintenance of strong synapses in adulthood. While the influence of the ovary is most likely due to estrogen secretion, the pattern of estrogen secretion required for maturation of the synapse in females is not known. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 441–448, 1998     

Facilitation at the sexually differentiated laryngeal synapse of Xenopus laevis

Under physiological conditions, the laryngeal synapse of male Xenopus laevis exhibits marked facilitation during repetitive nerve stimulation. The male laryngeal synapse is weak and requires facilitation to produce muscle action potentials and ultimately sound. The female laryngeal synapse is strong: muscle contractions are produced to single nerve stimuli. We sought to determine if laryngeal synapses of males and females also differ in their ability to facilitate. To measure facilitation, laryngeal muscle action potentials were suppressed either postsynaptically by bathing the preparation in saline containing curare or presynaptically by bathing the preparation in reduced calcium/elevated magnesium saline. Facilitation of postsynaptic potential amplitude or quantal content in response to paired pulses was measured in male and female larynges: there is no sex difference in paired pulse facilitation. Facilitation in response to trains of stimuli, in curare-blocked preparations, increased and reached plateau values more rapidly in females than in males, although the facilitation between the last and first pulses in the train was the same in the sexes. Thus, the sexually differentiated behavior of this synapse is controlled more by a sex difference in synaptic strength than by a sex difference in the ability to facilitate. Accepted: 14 June 1997     

The importance of conspecific facilitation during recruitment and regeneration: A case study in degraded mangroves

Facilitation can affect positively the survival, growth and fitness of neighboring plants and is able to promote the establishment of plant species under harsh environmental conditions. This study is one of the few to focus on conspecific nurse plants enabling the establishment of tree followers and the generally unknown interplay between facilitation and competition during recruitment and establishment.Field studies were conducted in an hypersaline area in north Brazil where the hydrological regime was disturbed by road construction in 1974. Avicennia germinans shrubs were able to recolonize subareas.Three different stages of re-colonization were identified by satellite imagery based on the vegetation coverage and were defined as the early, middle, and late stages. Different plant parameters, including height and crown radius of individual trees, were measured, and all plants separating mangrove seedlings and larger plants were stem-mapped.The Hegyi index was adapted to measure local interaction intensity, considering both positive (facilitation) and negative (competition) interactions among neighboring plants. Spatial point pattern analyses were combined with the interaction index to obtain a better overview of the strength and importance of the plant interactions within the different recolonizing stages.The spatial patterns displayed aggregation for all plants in all developmental stages. This is supported by the interaction index calculated for seedlings. The index, however, provides an early signal for the switching of plant interactions from facilitation to competition as the developmental stage increased. This feature remains undetected in the spatial patterns because self-thinning processes are linked to individual growth processes and require more time to take place.This study broadens the ecological concept of nurse plants from hetero- to conspecific, including the importance and strength of plant interactions. The studied degraded areas are recolonizing naturally, and conclusions can be drawn for restoration projects, which usually do not take facilitation effects into account.     

Augmentation and facilitation of transmitter release. A quantitative description at the frog neuromuscular junction

Endplate potentials were recorded from frog and toad sartorius neuromuscular junctions under conditions of greatly reduced quantal contents. The magnitudes of augmentation increased with the duration and frequency of stimulation, often increasing at an accelerating rate during 10-20-s conditioning trains. The magnitudes of the first and second components of facilitation also increased, but reached apparent steady state values within the first few seconds of stimulation. These observations could be accounted for by assuming (a) that augmentation and the first and second components of facilitation arise from underlying factors in the nerve terminal that act to increase transmitter release; (b) that each nerve impulse adds an increment to each of the underlying factors; (c) that the magnitude of the increment typically increases during the train for augmentation but remains constant for the components of facilitation; and (d) that the underlying factors decay with first-order kinetics with time constants of approximately 7 s for augmentation and 60 and 500 ms for the first and second components of facilitation, respectively. The increments of facilitation added by each impulse were about twice as large in the toad as in the frog. Facilitation was described better by assuming a power relationship between the underlying factor and the observed facilitation than by assuming a linear relationship. Augmentation was described by assuming either a linear or power relationship.     

Structural alterations at the neuromuscular junctions of matrix metalloproteinase 3 null mutant mice

Matrix metalloproteinases are important regulators of extracellular matrix molecules and cell-cell signaling. Antibodies to matrix metalloproteinase 3 (MMP3) recognize molecules at the frog neuromuscular junction, and MMP3 can remove agrin from synaptic basal lamina (VanSaun & Werle, 2000). To gain insight into the possible roles of MMP3 at the neuromuscular junction, detailed observations were made on the structure and function of the neuromuscular junctions in MMP3 null mutant mice. Striking differences were found in the appearance of the postsynaptic apparatus of MMP3 null mutant mice. Endplates had an increased volume of AChR stained regions within the endplate structure, leaving only small regions devoid of AChRs. Individual postsynaptic gutters were wider, containing prominent lines that represent the AChRs concentrated at the tops of the junctional folds. Electron microscopy revealed a dramatic increase in the number and size of the junctional folds, in addition to ectopically located junctional folds. Electrophysiological recordings revealed no change in quantal content or MEPP frequency, but there was an increase in MEPP rise time in a subset of endplates. No differences were observed in the rate or extent of developmental synapse elimination. In vitro cleavage experiments revealed that MMP3 directly cleaves agrin. Increased agrin immunofluorescence was observed at the neuromuscular junctions of MMP3 null mutant mice. These results provide strong evidence that MMP3 is involved in the control of synaptic structure at the neuromuscular junction and they support the hypothesis that MMP3 is involved in the regulation of agrin at the neuromuscular junction.     

Synaptic plasticity and humoral modulation of neuromuscular transmission in the lobster claw opener during the molt cycle

1. Neuromuscular properties of the lobster dactyl opener were studied at different stages of the molt cycle.2. Excitatory junctional potentials (EJPs) were found to be significantly larger in hard-shelled premolt lobsters than in soft-shelled postmolt animals.3. Inhibitory junctional potentials (IJPs) were larger in postmolt claw preparations than in premolt ones.4. Excitatory transmission was also monitored during superfusion with plasma samples obtained from lobsters in vartious stages of the molt cycle (postmolt = AB; intennolt = C; premolt = D₂).5. D₂ plasma increased EJP amplitude by an average of 28% relative to baseline levels (in saline), while AB plasma reduced EJP size by an average of 6%.6. Premolt and postmolt plasma produced opposite effects on quantal content (m); D₂; plasma increased m by a mean of 39%, whereas AB plasma caused a mean reduction by 10%, which suggests that humoral factors may act presynaptically to alter transmitter release.7. These results provide evidence of neuromuscular plasticity in the opener muscle during the molt cycle, and are consistent with changes in claw-opening behavior seen during this cycle.     

Identification of Most Stable Endogenous Control Genes for MicroRNA Quantification in the Developing Mouse Lung

MicroRNAs (miRNAs) are endogenous small non coding RNAs acting as negative regulators. miRNA are involved in lung development and pulmonary diseases. Measurement of their levels by qPCR is directly influenced by the stability of normalization gene(s), which can be affected by the experimental conditions. The developing lung is a changing tissue and one normalization gene showing stability on one developmental day may be modulated over time. Moreover, some developmental events are affected by sex, which also has to be considered. In this study, we compared stability of five putative control genes in the lung between sexes from the pseudoglandular to the alveolar stages and in adult lungs. Expression of sno135, sno142, sno202, sno234, and sno251 was studied by qPCR in male and female lung samples collected at seven time points from GD 15.5 to PN 30. Cq values of sno251 showed the highest variation across the different developmental stages, while sno234 was the most stable gene. Gene expression stability was studied by geNorm, NormFinder and BestKeeper. Our data showed that ranking of genes based on expression stability changed according to developmental time and sex. sno135/sno234 and sno142/sno234 were proposed as best combinations of normalization genes when both sexes and all the studied developmental stages are considered. Normalization of let7-a RNA levels with different pairs of control genes proposed by geNorm and NormFinder gave similar data, while the use of less stable genes introduced a statistically significant difference on PN 0. In conclusion, variations in stability of normalization gene expression are observed over time and according to sex during lung development. Best pairs of normalization genes are presented for specific developmental stages, and for the period extending from the pseudoglandular to the alveolar stages. The use of normalization genes selected for their expression stability is essential in lung development studies.     

Differential Responses of Crab Neuromuscular Synapses to Cesium Ion

Excitatory postsynaptic potentials (EPSP's) generated in crab muscle fibers by a single motor axon, differ in amplitude and facilitation. Some EPSP's are large at low frequencies of stimulation and show little facilitation; others are smaller and show pronounced facilitation. When K⁺ is replaced by Cs⁺ in the physiological solution, all EPSP's increase in amplitude, but small EPSP's increase proportionately more than large ones. Quantal content of transmission, determined by external recording at single synaptic regions, undergoes a much larger increase at facilitating synapses. The increase in quantal content of transmission is attributable to prolongation of the nerve terminal action potential in Cs⁺. After 1–2 h of Cs⁺ treatment, defacilitation of synaptic potentials occurs at synapses which initially showed facilitation. This indicates that Cs⁺ treatment drastically increases the fraction of the "immediately available" transmitter store released by each nerve impulse, especially at terminals with facilitating synapses. It is proposed that facilitating synapses normally release less of the "immediately available" store of transmitter than poorly facilitating synapses. Possible reasons for this difference in performance are discussed.     

Physiological studies during formation and development of rat neuromuscular junctions in tissue culture

Developing neuromuscular junctions in tissue cultures of rat embryo spinal cord and muscle were studied with intracellular recording. Excitatory junctional potentials (ejps) were found during nerve or spinal cord stimulation, and also arising spontaneously. The time-course of the potentials tended to be shorter in cultures older than 9 days in vitro than in more recently innervated cultures. Evidence of multiple innervation was found in many cells. In order to test the hypothesis that transmitter was released in integral multiples of a quantal amount and according to a Poisson distribution, mean quantum content was calculated from the coefficient of variation of ejp amplitudes, the percentage of zero responses, and the ratio of mean amplitude to mean of the smallest class of amplitudes. The three independent measures were in fair or good mutual agreement, implying that the mechanism of transmitter release in newly developed junctions is the same as in the adult. In newly formed junctions, ejps were subthreshold for action potential generation, but afterwards mean quantum content increased and action potentials were generated by single ejps. In fibers developing both with and without innervation, the entire muscle cell surface was as sensitive to acetylcholine as the adult end plate region. Innervation was related to cross-striation: every cross-striated fiber tested was found to be innervated, and denervation in vitro led to loss of distinct cross-striations.     

Synapse formation and modification between distal retinal neurons in larval and juvenile Xenopus

A serial section analysis of photoreceptor synaptic bases was undertaken in the clawed frog Xenopus laevis. The developmental period from tadpole stage 48 through metamorphosis was studied. Horizontal cells contacted rod and cone photoreceptors at ribbon synapses; the number of such contacts per receptor base was constant for rods, but increased for cones as a function of developmental stage. In pre-metamorphic animals bipolar cells contacted receptors only through basal junctions; their number in cone bases increased dramatically during development but was unchanged in rod bases. A densitometric estimation of the cleft width of basal junctions showed that it ranged from 10 to 18 nm, but the junctions could not be divided reliably into the 'wide' and 'narrow' categories reported for other vertebrate species. Near metamorphic climax a new type of ribbon-related bipolar cell junction appeared. Gap junctions between horizontal cells and conventional synapses of horizontal cell onto bipolar cell processes were first seen in mid-larval developmental stages.     

Biochemical studies of Aloe vera (Aloe barbadensis Miller) gel of the field grown and micropropagated plants at different developmental stages

The biochemical composition of leaf gel isolated from vegetative and micropropagated Aloe vera plants at different stages of development had been compared. The total sugars, fructose, sucrose and starch were higher in micropropagated leaf gel at all developmental stages. The micropropagated plants have higher α-amylase activity at all stages with maximum at 6 month old. Catalase and peroxidase activities were higher in younger leaves (up to 4 month old) in both types of plants. The calcium and magnesium level increases up to maturity and ranges from 0.19 to 1.12% and 0.02 to 0.10% respectively, in conventionally grown plants whereas it was a little higher in micropropagated plants. Micropropagated plants had 12.5% higher phosphorus levels. The micropropagated plants (1 month old) have 60% higher phenol content as compared to conventional plants. The micropropagated mature plants have 55 and 18.6% higher saponin and sterol contents as compared to conventional plants. The composition of aloe vera gel depends upon the growth stages and method of propagation.     

Mechanical rigidity and lignine content in different developmental phases of winter rape siliqua

The lignine content and mechanical rigidity of siliquae ofBrassica napus L. ssp.oleifera f.biennis, cv. T?ebí?ská were studied. Experimental plants were harvested in five consecutive periods and developmental stages of siliquae were determined on the basis of the weight of 1000 seeds. During the development of siliquae a gradual accumulation of lignine takes place; the content reached its maximum in certain developmental phases and then decreased. The mechanical rigidity of siliquae had a similar tendency. There were statistically significant differences in both properties studied within individual developmental phases. A highly significant positive correlation was found between lignine content and mechanical consistency.     

Microarray-based screening of the microRNAs associated with caryopsis development in Oryza sativa

Plant microRNAs modulate diverse developmental processes by regulating expression of their target genes. To explore potential miRNA-guided gene regulation in developing rice (Oryza sativa L.) caryopses, a miRNA microarray was used to identify miRNAs present at the different developmental stages. We found that 27 miRNAs, of which 16 were conserved miRNAs, were present in developing caryopses. High expression levels were detected for miR159, miR167, and miR530 at the morphogenesis stage and for miR169, miR435, and miR528 at the stage of accumulation of metabolites. Next, 26 target genes were predicted for seven of the detected miRNAs and the expression profiles of these miRNAs and their corresponding target genes were examined in developing caryopses. Our results suggest that the miRNAs and their target genes examined at the two distinct stages could contribute to the developmental progress of rice caryopses in concert with phytohormone signalling.     

Cytological and physiological changes related to cryotolerance in orthodox maize embryos during seed development

Cytological and physiological changes were studied in orthodox maize (Zea mays L.) embryos following the acquisition of cryotolerance to liquid nitrogen during seed development. It was found that the embryonic cells at radicle portion were hydrated at all stages investigated, but those at early stages contained fully functional organelles, which disappeared at last developmental stages, and reserve materials accumulated intensively during seed development. Total soluble sugar content in the embryos had a steady rise on fresh weight and moisture weight basis; meanwhile, soluble and heat-stable proteins increased progressively in their number and contents as embryos matured. These cytological and biochemical changes had good correspondence with acquisition of cryotolerance in maize embryos.     

Profiling of Volatile Compounds and Associated Gene Expression and Enzyme Activity during Fruit Development in Two Cucumber Cultivars

Changes in volatile content, as well as associated gene expression and enzyme activity in developing cucumber fruits were investigated in two Cucumis sativus L. lines (No. 26 and No. 14) that differ significantly in fruit flavor. Total volatile, six-carbon (C6) aldehyde, linolenic and linoleic acid content were higher during the early stages, whereas the nine-carbon (C9) aldehyde content was higher during the latter stages in both lines. Expression of C. sativus hydroperoxide lyase (CsHPL) mirrored 13-hydroperoxide lyase (13-HPL) enzyme activity in variety No. 26, whereas CsHPL expression was correlated with 9-hydroperoxide lyase (9-HPL) enzyme activity in cultivar No. 14. 13-HPL activity decreased significantly, while LOX (lipoxygenase) and 9-HPL activity increased along with fruit ripening in both lines, which accounted for the higher C6 and C9 aldehyde content at 0-6 day post anthesis (dpa) and 9-12 dpa, respectively. Volatile compounds from fruits at five developmental stages were analyzed by principal component analysis (PCA), and heatmaps of volatile content, gene expression and enzyme activity were constructed.