Synapse elimination from the mouse neuromuscular junction in vitro: A non-hebbian activity-dependent process

The effect of action potentials on elimination of mouse neuromuscular junctions (NMJ) was studied in a three compartment cell culture preparation. Axons from superior cervical ganglion or ventral spinal cord neurons in two lateral compartments formed multiple neuromuscular junctions with muscle cells in a central compartment. The loss of synapses over a 2–7-day period was determined by serial electrophysiological recording and a functional assay. Electrical stimulation of axons from one side compartment during this period, using 30-Hz bursts of 2-s duration, repeated at 10-s intervals, caused a significant increase in synapse elimination compared to unstimulated cultures (p< 0.001). The extent of homosynaptic and heterosynaptic elimination was comparable, i. e., of the 226 functional synapses of each type studied, 111 (49%) of the synapses that had been stimulated were eliminated, and 87 (39%) of unstimulated synapses on the same muscle cells were eliminated. Also, simultaneous bilateral stimulation caused significantly greater elimination of synapses than unilateral stimulation (p< 0.005). These observations are contrary to the Hebbian hypothesis of synaptic plasticity. A spatial effect of stimulus-induced synapse elimination was also evident following simultaneous bilateral stimulation. Prior to stimulation, most muscle cells were innervated by axons from both side compartments, but after bilateral stimulation, muscle cells were predominantly unilaterally innervated by axons from the closer compartment. These experiments suggest that synapse elimination at the NMJ is an activity-dependent process, but it does not follow Hebbian or anti-Hebbian rules of synaptic plasticity. Rather, elimination is a consequence of postsynaptic activation and a function of location of the muscle cell relative to the neuron. An interaction between spatial and activity-dependent effects on synapse elimination could help produce optimal refinement of synaptic connections during postnatal development. © 1993 John Wiley & Sons, Inc.     

A comparative study of physiological and structural changes at the myoneural junction in two species of frog after transection of the motor nerve

Summary Structural and functional behaviour of motor end-plates after transection of the motor nerve has been studied in two species of frog: Rana esculenta and Rana temporaria. The physiological results show that in both species there is a transient cessation of spontaneous activity followed by a resumption of miniature end-plate potentials (min. e.p.p.s.) after denervation. The characteristics of these potentials (frequency, distribution of amplitudes, time-course) are similar in the two species. However, some differences have been observed: Firstly, the period of silence lasts for 2–4 days in the case of Rana temporaria whereas it is prolonged to about 15 days in Rana esculenta. Secondly, the resumption of min. e.p.p.s. is gradual and after the 10th day of denervation remains constant in Rana temporaria. It is inconstant independent of the period of denervation in Rana esculenta. The morphological results show that the Schwann cell is constantly in contact with the post-synaptic membrane after about 6 days of denervation in both species. It is suggested that either the Schwann cell is capable of functioning for a limited period of time in Rana esculenta or is activated to produce min. e.p.p.s. only in certain cases.     

Genetic influences on mouse sperm capacitation in vivo and in vitro

Intial in vivo studies were performed to observe the proportion of eggs fertillized at specific intervals after natural mating and ovulation in our research mouse colony. Proestrous females of the C57BL/10Wt, SJL/Wt inbred strains and the F₁ hybrid cross (B10 × SJL or reciprocals) were paired in the after-noon with males of their respective strain and examined for vaginal plugs at the midpoint of the dark period (2400 hours). Oviducts were periodically collected from mated females, and ovulation was first observed at 4, 5.2, and 3 hours after 2400 hours in the B10, SJL, and F₁ hyrid, respectively. The clutch of eggs from each ovulating female, was placed in culture, and cleavage oviduct lavage verifying female mating was placed in culture, and cleavage was used as the criterion for fertilizaition. Fifty percent of the eggs were fertilized 2.2, 5.0, and 2.5 hours after ovulation in B10, SJL, and F₁ hybrid females, respectively. Because twice the legth of time was required to fertilize a similar proportion of eggs from the SJL strain as the F₁ hybrid, these two strains were used for determining their rate of fertilization under more fully controlled conditions in vitro. Forty-nine percent of F₁ hybrid eggs were fertilized after 4 hours incubation with SJL epididymal sperm, whereas 53% fo SJL and 56% of F₁ hybrid eggs were fertilized after only 2 hours incubation with F₁ hybrid epididymal sperm. Thus, using sperm from these two mouse strains, the amount of time required to fertilize approximately 50% of the eggs within a clutch both in vivo and vitro was very similar. These observations demonstrte teh validity of using this in vitro system for fertilization studies and confirm that the temporal events in sperm capacitation and egg penetration are dependent on the genotype of the sperm. Similarities in fertilization rates at specific times after ovulation or insemination in vitro imply that the initiationof sperm capacitation in vivo occurs near the time of ovulation and several hours after mating. We tentatively suggest that follicular fluid may be required for completion of mouse sperm capacitaiton in vivo.     

A comparative study on wound-healing in neonatal and adult mouse epidermis in vivo

Abstract. A cut made into the back skin of either newborn or adult mice evokes, at both ages, a hyperproliferative response in the epidermis. Differences in the reaction of neonatal as compared with adult epidermis are found in the spatial distribution of proliferative activity as well as in its time course. The response in adult mouse epidermis is inhibited by local application of indomethacin, whereas the response of the newborn epidermis is not.     

ER stress responses in the absence of apoptosome: A comparative study in CASP9 proficient vs deficient mouse embryonic fibroblasts

Cells respond to endoplasmic reticulum (ER) stress through the unfolded protein response (UPR), autophagy and cell death. In this study we utilized casp9^+/+ and casp9^−/− MEFs to determine the effect of inhibition of mitochondrial apoptosis pathway on ER stress-induced-cell death, UPR and autophagy. We observed prolonged activation of UPR and autophagy in casp9^−/− cells as compared with casp9^+/+ MEFs, which displayed transient activation of both pathways. Furthermore we showed that while casp9^−/− MEFs were resistant to ER stress, prolonged exposure led to the activation of a non-canonical, caspase-mediated mode of cell death.     

A transmission electron microscope study using vitrified ice sections of predentin: structural changes in the dentin collagenous matrix prior to mineralization

The assembly of the collagenous organic matrix prior to mineralization is a key step in the formation of bones and teeth. This process was studied in the predentin of continuously forming rat incisors, using unstained vitrified ice sections examined in the transmission electron microscope. Progressing from the odontoblast surface to the mineralization front, the collagen fibrils thicken to ultimately form a dense network, and their repeat D-spacings and banding patterns vary. Using immunolocalization, the most abundant noncollagenous protein in dentin, phosphophoryn, was mapped to the boundaries between the gap and overlap zones along the fibrils nearest the mineralization front. It thus appears that the premineralized collagen matrix undergoes dynamic changes in its structure. These may be mediated by the addition and interaction with the highly anionic noncollagenous proteins associated with collagen. These changes presumably create a collagenous framework that is able to mineralize.     

A comparative study of primary and secondary granules in monocytopoiesis and myelopoiesis of mouse bone marrow

Summary The differentiation and maturation of monocytes and neutrophil granulocytes were studied in bone marrow of normal mice by electron microscopy and cytochemical assessment of peroxidatic activity. The granule populations of the mature cells of bone marrow were identified and investigated to obtain a basis for the analysis of the earlier stages of maturation. Mature monocytes and neutrophils showed primary and secondary granules, and mature neutrophils had more of both kinds. The size, shape, and number of primary granules proved to offer the most reliable criteria for distinguishing promonocytes and promyelocytes. The primary granules of monocytes were smaller than those of mature neutrophils and were either spherical (smallest diameter 50–200 nm) or elongate (100×400 nm). Both granules had a homogeneous matrix. The granules of the granulocytes were either spherical (smallest diameter 200–300 nm) or elongate (150–200×300–500 nm), and some of them had a crystalline inclusion.     

EGF induces efficient Cx43 gap junction endocytosis in mouse embryonic stem cell colonies via phosphorylation of Ser262, Ser279/282, and Ser368

Gap junctions (GJs) traverse apposing membranes of neighboring cells to mediate intercellular communication by passive diffusion of signaling molecules. We have shown previously that cells endocytose GJs utilizing the clathrin machinery. Endocytosis generates cytoplasmic double-membrane vesicles termed annular gap junctions or connexosomes. However, the signaling pathways and protein modifications that trigger GJ endocytosis are largely unknown. Treating mouse embryonic stem cell colonies – endogenously expressing the GJ protein connexin43 (Cx43) – with epidermal growth factor (EGF) inhibited intercellular communication by 64% and activated both, MAPK and PKC signaling cascades to phosphorylate Cx43 on serines 262, 279/282, and 368. Upon EGF treatment Cx43 phosphorylation transiently increased up to 4-fold and induced efficient (66.4%) GJ endocytosis as evidenced by a 5.9-fold increase in Cx43/clathrin co-precipitation.     

A potential role for NEDD1 and the centrosome in senescence of mouse embryonic fibroblasts

Mouse embryonic fibroblasts (MEFs) are commonly grown in cell culture and are known to enter senescence after a low number of passages as a result of oxidative stress. Oxidative stress has also been suggested to promote centrosome disruption; however, the contribution of this organelle to senescence is poorly understood. Therefore, this study aimed to assess the role of the centrosome in oxidative stress induced-senescence using MEFs as a model. We demonstrate here that coincident with the entry of late-passage MEFs into senescence, there was an increase in supernumerary centrosomes, most likely due to centrosome fragmentation. In addition, disrupting the centrosome in early-passage MEFs by depletion of neural precursor cell expressed developmentally downregulated gene 1 (NEDD1) also resulted in centrosomal fragmentation and subsequent premature entry into senescence. These data show that a loss of centrosomal integrity may contribute to the entry of MEFs into senescence in culture, and that centrosomal disruption can cause senescence.     

Effect of antibody to detergent solubilized zonae pellucidae on in vivo and in vitro fertilization in the mouse

An antiserum was produced in one rabbit against mouse zonae pellucidae solubilized with 70 mM Na₂SO₃, 1% SDS, and 0.04 mM CuSO₄. An IgG of zona antibody completely inhibited fertilization both in vitro and in vivo in the mouse. F(ab′)₂ fragment obtained by pepsin digestion of IgG zona antibody inhibited fertilizability of eggs in vitro but did not inhibit fertilization in vivo after passive immunization.     

A comparative study of eicosapentaenoic acid metabolism by human platelets in vivo and in vitro

During long-term dietary n-3 fatty acid supplementation, eicosapentaenoic acid (EPA) is not incorporated into phosphatidylinositol or -serine of human platelets in vivo and is not detectable in phosphatidic acid upon stimulation with thrombin. However, EPA is released from platelet phospholipids and metabolized to thromboxane B3 (TXB3). In contrast, in vitro, platelets incorporate [14C]EPA into phosphatidylinositol, whether they contain endogenous EPA in their cellular lipids or not. Following platelet stimulation, [14C]EPA appears in phosphatidic acid, as free fatty acid, and is transformed to TXB3. We conclude that the fatty acid compositions of platelet phospholipid subclasses are regulated with a high degree of specificity in vivo. Qualitative differences exist between in vivo and in vitro uptake of EPA into platelet phospholipid subclasses. After in vivo incorporation, EPA is released by action of a phospholipase A2.     

In vivo and in vitro growth of Mycobacterium marinum at homoeothermic temperatures

Mycobacterium marinum can cause systemic infection in fishes and skin infection in humans. Most strains grow better at <37 degrees C, which can explain the rarity of infections in humans. The ability of strains from humans and fish to grow in various conditions, and in macrophages from carp, humans, and mouse was evaluated, as was the ability of the three fish isolates to infect mice. Significant differences of growth in vitro and in vivo were observed. All fish strains caused both footpad and deep tissue infections, and two, which grew very poorly or not all at 37 degrees C, proliferated in mammalian macrophages.     

Epithelial differentiation at the mucogingival junction: A stereological comparison of the epithelia of the vestibular gingiva and alveolar mucosa

Summary The epithelial lining of normal human vestibular gingiva and the adjoining alveolar mucosa was subjected to a comparative stereological analysis. Five biopsies collected from 11 to 12 year-old males and females were selected from a total of 14 specimens and, under standardized conditions, processed for light- and electron microscopy. At two levels of magnification, electron micrographs were sampled from five strata in the oral-gingival, and from four strata in the alveolar-mucosal epithelium, mostly in regions of epithelial ridges. Standardized sterological point counting techniques were employed to analyze a total of 710 and 540 electron micrographs from the oralgingival and the alveolar-mucosal epithelium, respectively. The two epithelia, although of similar thickness, show different differentiation patterns. The oral-gingival epithelium consists of four cytologically different strata, the major differentiation step occurring between the lower and upper stratum spinosum of epithelial ridges. Standardized stereological point counting techniques were alveolar-mucosal epithelium, consisting of two cytologically different cell compartments, displays a broad, superficial zone of differentiated flat cells, with 60% of the cytoplasm filled with a dense network of cytoplasmic filaments. The major differentiation step occurs between basal and lower spinous layers. Differentiation phenomena in both epithelia are discussed and individual variations are interpreted in view of genetically determined factors.

---

Zusammenfassung Das Epithel der normalen menschlichen vestibulären Gingiva und der benachbarten Alveolarschleimhaut wurde vergleichend stereologisch analysiert. Fünf Biopsien von 11–12 Jahre alten gesunden Knaben und Mädchen, die aus insgesamt 14 Biopsien ausgewählt worden waren, wurden standardisiert für licht- und elektronenmikroskopische Studien verarbeitet. Elektronenmikroskopische Aufnahmen wurden in zwei Vergrößerungsstufen aus fünf Schichten des oralen Gingivaepithels und aus vier Schichten des Alveolarschleimhautepithels, zumeist im Bereich epithelialer Leisten, gewonnen. Insgesamt wurden 710 Bilder aus dem oralen Gingivaepithel und 540 Bilder aus dem Alveolarschleimhautepithel mit Hilfe von standardisierten stereologischen Punktzählverfahren analysiert. Die untersuchten Epithelien sind etwa gleich dick, weisen aber sehr verschiedenartige Differenzierungsmuster auf. Das orale Gingivaepithel besteht aus vier zytologisch unterschiedlichen Schichten und bildet ein parakeratinisiertes, 0,1 mm dickes Stratum corneum, wobei der Hauptdifferenzierungsschritt zwischen dem unteren und dem oberen Stratum spinosum im Bereich der epithelialen Leisten erfolgt. Das Alveolarschleimhautepithel weist zwei zytologisch unterschiedliche Zellkompartimente auf und bildet eine breite oberflächliche Lage flacher und differenzierter Zellen, deren Zytoplasma zu 60% aus einem dichten Maschenwerk zytoplasmatischer Filamente besteht. Der Hauptdifferenzierungsschritt dieses Epithels liegt zwischen dem Stratum basale und dem unteren Stratum spinosum. Die verschiedenen Differenzierungsvorgänge werden diskutiert und individuelle Variationen, die in beiden Epithelien auftreten, im Hinblick auf genetische Faktoren erklärt.

     

Initial reactions in the mineralization of 2-sulfobenzoate by Pseudomonas sp. RW611

Abstract Pseudomonas sp. strain RW611 utilized the ammonium salt of 2-sulfobenzoate as sole source of carbon, sulfur, nitrogen, and energy. The xenobiotic sulfo substituent was dioxygenolytically eliminated as sulfite, which was then slowly oxidized to sulfate. 2,3-Dihydroxybenzoate, which resulted from desulfonation underwent meta -cleavage, mediated by 2,3-dihydroxybenzoate 3,4-dioxygenase activity. This enzyme was inhibited by 3-chlorocatechol and 2,3,4-trihydroxybenzoate.     

Fertilization of cumulus-free,zona-intact mouse ova in vitro at high and low sperm concentrations

The effect of varying the sperm concentration between 2 × 10⁵ sperm/ml and 8 × 10⁶ sperm/ml on fertilization of cumulus-free, zona-intact F1 (CBA × C57BL) mouse ova by QS and F1 (CBA × C57BL) mouse spermatozoa was studied. The spermatozoa from both strains of mice exhibited optimal fertilization rates at 2 × 106 sperm/ml. However, at sperm concentrations greater than 4 × 106 sperm/ml and less than 1 × 106 sperm/ml, fertilization rates were significantly reduced. F1 spermatozoa were more susceptible to dilution than QS spermatozoa. A significant interaction between strain and sperm concentration indicated that the two strains produced different fertilization rates at different sperm densities. Extracts of epididymal fluid, medium from capacitated spermatozoa, or ampulla fluid did not improve the fertilization rate at 2 × 105 sperm/ml, but retaining the cumulus oophorus did. The decrease in fertilization rate at 8 × 106 sperm/ml can in part be attributed to a nondialysable inhibitor from the neat sperm preparation that appeared to be of epididymal origin.     

Inhibitors of in vitro mineralization from rabbit aorta and their role in biomineralization

Mineralization of aorta is known to occur late in life and appears to be a pathological phenomenon. In vitro studies revealed that the matrix prepared from the thoracic aorta pieces after their extraction with 3% Na₂HPO₄ and 0.1 mM CaCl₂ were mineralized under physiological conditions of temperature, pH, and ionic strength of the media to form matrix-bound mineral phase resembling hydroxyapatite in nature. However, the matrix identically prepared from the unextracted rabbits aortae failed to mineralize under identical assay conditions. The addition of the aorta extract in the assay system inhibited the above mineralization process. Standard biochemical techniques, e.g., dialysis, ion exchange, and molecular sieve chromatography, sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and amino acid analysis by high-performance liquid chromatography were employed to isolate, purify, and characterize the potent inhibitory biomolecules from the aorta extract. The inhibitory activity of the aorta extract was found to be primarily due to the presence of three biomolecules having molecular weights of 66, 45, and 27–29 kDa. The above inhibitory biomolecules loosely associated with aorta may be involved in the control of calcification associated with arteriosclerosis. J. Cell. Biochem. 68:287–297, 1998. © 1998 Wiley-Liss, Inc.     

A comparative study of apolipoproteins in mouse and rat

1. Apolipoproteins isolated from plasma samples of 10 inbred strains of mice and 17 inbred strains of rats were subjected to isoelectric focusing and second-dimension-pore-gradient-SDS-electrophoresis. 2. All major HDL apolipoproteins could be identified by their isoelectric point and mol. wt. 3. In inbred strains of mice polymorphism could be demonstrated for apo A-I and apo A-II. 4. In inbred strains of rats no apolipoprotein polymorphism could be demonstrated.