The regeneration of neuromuscular junctions during spontaneous re-innervation of the rat diaphragm

Summary Electron microscopic observations have been made on the regeneration of neuromuscular junctions during spontaneous re-innervation of the rat diaphragm, following unilateral transsection of the phrenic nerve. 3 and 4 weeks after denervation motor end plates displayed the pattern of almost complete degeneration, i.e. persisting subneural foldings, deprived of neural contact and covered with collagen fibrils and fibrocytes. From observations at 5, 10 and 24 weeks after denervation the following sequence of events could be established: a few small axon terminals, accompanied by Schwann cells, became apposed to subneural folds, while most foldings were covered initially by Schwann cells or still by collagen fibrils. Gradually an increasing number of subneural folds came into contact with axon terminals. At 24 weeks all junctions displayed the pattern of a mature motor end plate. In the majority of regenerating neuromuscular junctions single dense-cored vesicles of approximately 900–1200 Å were present in axon terminals.It is concluded that under the present conditions restoration of neuromuscular transmission is accomplished by a re-innervation of the preserved subneural apparatuses of former junctions by regenerating axons. The significance of the occurrence of dense-cored vesicles in regenerating motor end plates is discussed.This work was supported by the Deutsche Forschungsgemeinschaft and the Stiftung Volkswagenwerk.     

Immunolocalization of hepatic estrogen and progesterone receptors in the female lizard Uromastyx acanthinura

The hormonal regulation of hepatic synthesis of vitellogenin during the annual reproductive cycle was performed for the first time in the deserticole, oviparous, diurnal and herbivorous Uromastyx acanthinura, a lizard belonging to the Agamidae family. In order to elucidate what kind of estrogen receptor is involved in this process, an immunohistochemical study was performed. Changes were obtained in the labeling and cellular distribution of the estrogen and progesterone receptors according to the period of the reproductive cycle and the experimental administration of 17β-estradiol. Only the ERβ subtype was present; it was found in all phases of the cycle with a variable localization: nuclear and cytosolic during vitellogenesis, mainly cytosolic in the female with egg retention (luteal phase) and strictly cytosolic in females at sexual rest. The progesterone receptors were present only at the luteal phase and during sexual rest and disappeared completely from females after 17β-estradiol treatment in sexual rest. Our data suggested that mediation of action of the 17β-estradiol in the vitellogenin synthesis in the lizard U. acanthinura occured via ERβ. PRA and PRB could both be necessary for the negative effect of progesterone on the hepatic synthesis of vitellogenin.     

Ethylene and shoot regeneration: hookless1 modulates de novo shoot organogenesis in Arabidopsis thaliana

We have investigated the role of ethylene in shoot regeneration from cotyledon explants of Arabidopsis thaliana. We examined the ethylene sensitivity of five ecotypes representing both poor and prolific shoot regenerators and identified Dijon-G, a poor regenerator, as an ecotype with dramatically enhanced ethylene sensitivity. However, inhibiting ethylene action with silver nitrate generally reduced shoot organogenesis in ecotypes capable of regeneration. In ecotype Col-0, we found that ethylene-insensitive mutants (etr1-1, ein2-1, ein4, ein7) exhibited reduced shoot regeneration rates, whereas constitutive ethylene response mutants (ctr1-1, ctr1-12) increased the proportion of explants producing shoots. Our experiments with ethylene over-production mutants (eto1, eto2 and eto3) indicate that the ethylene biosynthesis inhibitor gene, ETO1, can act as an inhibitor of shoot regeneration. Pharmacological elevation of ethylene levels was also found to significantly increase the proportion of explants regenerating shoots. We determined that the hookless1 (hls1-1) mutant, a suppressor of the ethylene response phenotypes of ctr1 and eto1 mutants, is capable of dramatically enhancing shoot organogenesis. The effects of ACC and loss of HLS1 function on shoot organogenesis were found to be largely additive.     

The effect of temperature on digestive and assimilation efficiency,gut passage time and appetite in an ambush foraging lizard,<Emphasis Type="Italic"> Cordylus melanotus melanotus</Emphasis>

In ectotherms, an increase in body temperature increases metabolic rate and may increase rates of digestive processes. We measured the thermal dependence of the apparent digestive and apparent assimilation efficiencies (ADE and AAE), gut passage time (GP) and appetite in Cordylus melanotus melanotus, a medium sized Crag Lizard, which is endemic to South Africa. Trials were conducted at 20, 22, 25, 30, 32 and 35 °C under controlled conditions. Trials lasted 14 days, during which, lizards were fed ca. 1 g mealworms per day. Glass beads were used as markers to determine GP at the beginning and end of trials. Faeces and urates were collected daily and oven dried at 50 °C. The energy content of egested matter was then measured using bomb calorimetry. ADE and AAE were not affected by temperature for either males or females. The mean±SE ADE and AAE were 94.4±0.3% and 87.2±0.6%, respectively. GP was not significantly different between males and females at any temperature, but decreased significantly with increasing temperature. Appetite was significantly different between the different temperatures measured. The decrease of gut passage time with increasing temperature was expected, since the digestive and assimilation efficiencies are similar over the range of temperatures tested. Lizards are thus assimilating a similar proportion of ingested energy, but at faster rates at higher temperatures. The results indicate that the digestive physiology of this species results in maximum energy gain per meal in environments where food is scarce.Abbreviations AAE apparent assimilation efficiency - ADE apparent digestive efficiency - AE assimilation efficiency - DE digestive efficiency - GP gut passage rate - NEA net energy absorbed through gut - NER net usable energy retained - SVL snout-vent length - T _b body temperature Communicated by G. Heldmaier     

Structure and activation of MuSK,a receptor tyrosine kinase central to neuromuscular junction formation

MuSK (muscle-specific kinase) is a receptor tyrosine kinase that plays a central signaling role in the formation of neuromuscular junctions (NMJs). MuSK is activated in a complex spatio-temporal manner to cluster acetylcholine receptors on the postsynaptic (muscle) side of the synapse and to induce differentiation of the nerve terminal on the presynaptic side. The ligand for MuSK is LRP4 (low-density lipoprotein receptor-related protein-4), a transmembrane protein in muscle, whose binding affinity for MuSK is potentiated by agrin, a neuronally derived heparan-sulfate proteoglycan. In addition, Dok7, a cytoplasmic adaptor protein, is also required for MuSK activation in vivo. This review focuses on the physical interplay between these proteins and MuSK for activation and downstream signaling, which culminates in NMJ formation. This article is part of a Special Issue entitled: Emerging recognition and activation mechanisms of receptor tyrosine kinases.     

Abnormal development of the neuromuscular junction in Nedd4-deficient mice

Nedd4 (neural precursor cell expressed developmentally down-regulated gene 4) is an E3 ubiquitin ligase highly conserved from yeast to humans. The expression of Nedd4 is developmentally down-regulated in the mammalian nervous system, but the role of Nedd4 in mammalian neural development remains poorly understood. Here we show that a null mutation of Nedd4 in mice leads to perinatal lethality: mutant mice were stillborn and many of them died in utero before birth (between E15.5-E18.5). In Nedd4 mutant embryos, skeletal muscle fiber sizes and motoneuron numbers are significantly reduced. Surviving motoneurons project axons to their target muscles on schedule, but motor nerves defasciculate upon reaching the muscle surface, suggesting that Nedd4 plays a critical role in fine-tuning the interaction between the nerve and the muscle. Electrophysiological analyses of the neuromuscular junction (NMJ) demonstrate an increased spontaneous miniature endplate potential (mEPP) frequency in Nedd4 mutants. However, the mutant neuromuscular synapses are less responsive to membrane depolarization, compared to the wildtypes. Ultrastructural analyses further reveal that the pre-synaptic nerve terminal branches at the NMJs of Nedd4 mutants are increased in number, but decreased in diameter compared to the wildtypes. These ultrastructural changes are consistent with functional alternation of the NMJs in Nedd4 mutants. Unexpectedly, Nedd4 is not expressed in motoneurons, but is highly expressed in skeletal muscles and Schwann cells. Together, these results demonstrate that Nedd4 is involved in regulating the formation and function of the NMJs through non-cell autonomous mechanisms.     

Temporal distribution of 5BrdU-labelled cells suggests that most injured tissues contribute proliferating cells for the regeneration of the tail and limb in lizard

After tail and limb amputation in lizard, injection of 5BrdU for 6 days produces immunolabelled cells in most tissues of tail and limb stumps. After further 8 and 16 days, and 14 and 22 days of regeneration, numerous 5BrdU-labelled cells are detected in regenerating tail and limb, derived from most stump tissues. In tail blastema cone at 14 days, sparse-labelled cells remain in proximal dermis, muscles, cartilaginous tube and external layers of wound epidermis but are numerous in the blastema. In apical regions at 22 days of regeneration, labelled mesenchymal cells are sparse, while the apical wound epidermis contains numerous labelled cells in suprabasal and external layers, indicating cell accumulation from more proximal epidermis. Cell proliferation dilutes the label, and keratinocytes take 8 days to migrate into corneous layers. In healing limbs, labelled cells remain sparse from 14 to 22 days of regeneration in wound epidermis and repairing tissues and little labelling dilution occurs indicating low cell proliferation for local tissue repair but not distal growth. Labelled cells are present in epidermis, intermuscle and peri-nerve connectives, bone periosteum, cartilaginous callus and sparse fibroblasts, leading to the formation of a scarring outgrowth. Resident stem cells and dedifferentiation occur when stump tissues are damaged.     

Effect of castration and testosterone administration on the neuromuscular junction in the levator ani muscle of the rat

Summary The ultrastructure of the neuromuscular junction (n.m.j.) of the androgen-sensitive levator ani muscle was studied in normal adult male rats, in 8-month-old rats castrated at the age of one month and in castrated rats treated with testosterone propionate (TP). Castration does not result in significant changes of the n.m.j. The density of synaptic vesicles and the postsynaptic junctional folds remain practically normal in spite of marked atrophy of the muscle. TP administration for 7 days results in marked changes in preand postsynaptic structures. There is slow progressive depletion of synaptic vesicles, appearance of cisternae and coated vesicles in axon terminals, and coalescence of coated vesicles with the plasma membrane. Coated vesicles are also found inside Schwann cells and among junctional folds. Dense core vesicles appear both in the axon terminals and in the postsynaptic area. Collateral sprouting of terminal axons with the formation of new immature junctions is observed. After 35 days of TP administration depletion of synaptic vesicles continues. Glycogen -particles, mostly freely dispersed, occasionally seen in axon terminals 7 days after TP administration, subsequently increase in number. In the endplate zone of the muscle fibre increased protein synthesis is indicated by a rapid increase in ribosomes and irregularly located myofilaments and myofibrils. The appearance of n.m.j. after testosterone administration resembles that described after nerve stimulation; the degree of change is however less pronounced.The authors wish to acknowledge the skillful technical assistance of Mrs. L. Vedralová     

Changes in the distribution and size of synaptic vesicles in neuromuscular junctions of Locusta migratoria after stimulation and rest

Summary The distribution and size of synaptic vesicles in excitatory terminals of the extensor tibiae muscle were determined after stimulation at frequencies varying from 0.5 to 100 Hz and after a subsequent rest. Only in preparations stimulated at the higher frequencies do these parameters differ from those in the controls. The synaptic vesicles in the nonsynaptic areas of these terminals are depleted in number, and the remaining vesicles are reduced in size. These effects are reversed after a 1 h rest.The authors thank Mr. M.J. Parker for invaluable help with the statistical analyses. R.P. Botham thanks the S.R.C. for financial assistance     

In vitro studies of the influence of prolactin on tail regeneration in the adult newtNotophthalmus viridescens

Summary In vitro experiments were carried out to determine the effects of prolactin, and prolactin in combination with other hormones on the regeneration of adult newt tail blastemata. A total of 271 blastemata were explanted 13 days postamputation and were organ cultured for 96 h at 20 (±1)°C. Treatment with prolactin alone resulted in an increase in the blastema cell density of the tail regenerates. Cell accumulation and cell alignment were observed ventral to the reconstituted spinal cord. Prolactin and thyroxine, in combination, improved development of tail regenerates as compared with treatment with prolactin or thyroxine singly, supporting the results of earlier in vivo studies. Optimal development was obtained only when prolactin, insulin, thyroxine and hydrocortisone were added to the culture medium. Regeneration of tail explants maintained in medium augmented with the four hormones closely resembles that of in vivo tail blastemata 17 days post-amputation.Supported by grant A-1208 from the Natural Sciences and Engineering Research Council of Canada to R.A.L.     

The interactions of ouabain with post-tetanic and facilitatory drug potentiations at cat soleus neuromuscular junctions in vivo

Cat soleus motor nerve terminals, after high frequency conditioning, generate a post-tetanic repetition (PTR) which leads to a post-tetanic (PTP) of the muscle response. This property enables quantitative assessment of enhancement or depression of this nerve terminal excitability in vivo. The present study focuses on ionic mechanisms underlying the PTRs produced in this neuromuscular system either by high frequency stimulation or edrophonium. Ouabain was used as a specific probe for inhibition of Na⁺–K⁺ ATPase and its known consequences on Na⁺ and Ca²⁺ translocation. Ouabain pretreatment doubled the duration over which single stimuli, following either high frequency or edrophonium conditioning produced PTR. Ouabain in the doses used had no effectper se but as a function of dose augmented the frequency dependent responses. This pointed to Na⁺ loading of nerve terminals via high frequency stimulation plus ouabain inhibition of Na⁺–K⁺ ATPase. Ouabain potentiation of PTR responses evidently depends on exchange of intra-terminal sodium for external calcium. Thus, calcium entry blockers, Mn²⁺, and Co²⁺ suppressed or abolished the potentiations both before and after ouabain. Diphenylhydantoin, a Na⁺ and Ca²⁺ blocker, acted similarly. The effects of stimulation frequency, ouabain and the sequence of events leading to PTR in the soleus neuromuscular system appeared in general no different from those derived from the many in vitro microphysiologic studies of this phenomenon. Thus, EPPs were augmented and prolonged. It was concluded that intracellular Ca²⁺ is critical for regulating the stability of systems in which repetitive firing is both a normal and abnormal function.Special issue dedicated to Dr. Sidney Udenfriend     

Genetic variation and de novo mutations in the parthenogenetic Caucasian rock lizard Darevskia unisexualis

Unisexual all-female lizards of the genus Darevskia that are well adapted to various habitats are known to reproduce normally by true parthenogenesis. Although they consist of unisexual lineages and lack effective genetic recombination, they are characterized by some level of genetic polymorphism. To reveal the mutational contribution to overall genetic variability, the most straightforward and conclusive way is the direct detection of mutation events in pedigree genotyping. Earlier we selected from genomic library of D. unisexualis two polymorphic microsatellite containing loci Du281 and Du215. In this study, these two loci were analyzed to detect possible de novo mutations in 168 parthenogenetic offspring of 49 D. unisexualis mothers and in 147 offspring of 50 D. armeniaca mothers. No mutant alleles were detected in D. armeniaca offspring at both loci, and in D. unisexualis offspring at the Du215 locus. There were a total of seven mutational events in the germ lines of four of the 49 D. unisexualis mothers at the Du281 locus, yielding the mutation rate of 0.1428 events per germ line tissue. Sequencing of the mutant alleles has shown that most mutations occur via deletion or insertion of single microsatellite repeat being identical in all offspring of the family. This indicates that such mutations emerge at the early stages of embryogenesis. In this study we characterized single highly unstable (GATA)(n) containing locus in parthenogenetic lizard species D. unisexualis. Besides, we characterized various types of mutant alleles of this locus found in the D. unisexualis offspring of the first generation. Our data has shown that microsatellite mutations at highly unstable loci can make a significant contribution to population variability of parthenogenetic lizards.     

Differential Expression of Ciliary Neurotrophic Factor Receptor in Skeletal Muscle of Chick and Rat After Nerve Injury

Abstract: The activities of ciliary neurotrophic factor (CNTF) were initially thought to be restricted to cells in the nervous system. However, the recent identification of its receptor specificity-conferring α component (CNTFRα) in skeletal muscle has provided the clue to the unexpected actions of CNTF in the periphery. In the present study, we demonstrated that the mRNA expression of CNTFRα in chick skeletal muscle was decreased by ∼10-fold after nerve transection; this finding is in sharp contrast to the dramatic up-regulation observed in denervated rat muscle. As a first step toward investigating the differential regulation of CNTFRα in chick and rat, we examined the mRNA expression of CNTFRα in different types of muscle following nerve injury in young and adult animals. Our findings demonstrated that the differential expression of CNTFRα observed in denervated skeletal muscle of the chick and rat was not dependent on age or muscle type. The temporal profile of the changes in CNTFRα expression was, however, dependent on the age of the chick as well as the types of muscle. Furthermore, the low level of CNTFRα expression observed in denervated chick muscle recovered to almost control levels in regenerating skeletal muscle. Taken together, our findings provided the first extensive analysis on the mRNA expression of CNTFRα and the α subunit of the acetylcholine receptor in various skeletal muscles of the chick following nerve injury and regeneration.     

The role of the activin-follistatin system in the developmental and regeneration processes of the kidney

Regeneration processes in many tissues are modulated by various factors, which are involved in their organogenesis. Activin A, a member of the TGF-β superfamily, inhibits branching tubulogenesis of the kidney in organ culture system as well as in in vitro tubulogenesis model. On the other hand, follistatin, an antagonist activin A, reverses the effect of activin A on kidney development, induces branching tubulogenesis, and also promotes tubular regeneration after ischemia/reperfusion injury by blocking the action of endogenous activin A. The activin-follistatin system is one of the important regulatory systems modulating developmental and regeneration processes of the kidneys.     

Ultrastructure and immunocytochemistry of neurons in the supraoptic and paraventricular nuclei of the lizard Liolaemus cyanogaster

Summary The supraoptic (SON) and paraventricular (PVN) nuclei of the lizard Liolaemus cyanogaster c. were studied by use of histochemical, immunocyto-chemical and electron microscopic methods. The immunofluorescence staining for neurophysin was applied to methacrylate-embedded material before and after treatment of the sections with urea and trypsin. Pseudoisocyanine was applied to sections previously used for immunocytochemistry. The ultrastructural study showed that the SON and PVN neurons possess neurosecretory granules (nsg), distributed throughout the perikaryon, and large (2 to 12 m) electron-dense droplets located within dilatations of the cisternae of the rough endoplasmic reticulum. Whereas the perikaryon (nsg) and the secretory droplets are stainable with pseudoisocyanine, only the former displays immunoreactivity for neurophysin. However, after treating the sections with urea and trypsin, the same secretory droplets become immunoreactive. It is suggested that the secretory droplets are sites of storage for the precursor of neurophysin, and that the tryptic digestion either triggers its conversion into neurophysin or exposes its immunoreactive sites. Based on the ultrastructure and the histochemical behavior of the secretory droplets, it is also postulated that they contain, in addition to peptides, a glycoprotein component.Supported by Grant S-77-28 from the Dirección de Investigaciones, Universidad Austral de ChileThe authors wish to thank Prof. B.T. Pickering for providing the antineurophysin serum and Mrs. Elizabeth Santibáñez for her assistance