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Results for “Microinjection”

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PubMed1998

[Effects of glucocorticoids on cardiovascular response after microinjection of noradrenaline/neuropeptide Y into nucleus tractus solitarius and the underlying mechanism in rats].

The present study was designed to observe the effect of glucocorticoids in the NTS on cardiovascular regulation, and to explore the effect of glucocorticoids on cardiovascular responses to microinjection of NA/NPY into the NTS. A large dosage of Dex was found to evoke a decrease of blood pressure and heart rate, and an increase of serum NO concentration. A small dosage of Dex rapidly attenuated or even abolished the cardiovascular responses to NA/NPY microinjected into NTS, and this effect continued for a longer period. It indicates that the effect of Dex on cardiovascular responses to NA/NPY is mediated through both the genomic and non-genomic pathways. As regards the non-genomic mechanism, it was found that the rapid inhibitory effect had nothing to do with the intracellular glucocorticoid receptors, but was mediated through the stimulation of GABAA receptor to decrease the depressor reflex, or decreasing the sensitivity of alpha 2-adrenergic receptor and inhibiting NO formation, or directly affecting the potassium and calcium channel in the membrane and altering the response of cardiovascular neurons in the NTS to NA/NPY.

Animals↗

PubMed1991

Loss of heterozygosity for the short arm of chromosome 11 (11p15) in human milk epithelial cells immortalized by microinjection of SV40 DNA.

We have developed, by microinjection of SV40 DNA into human milk epithelial cells, a new mammary cell line, Hu-MI, which exhibits the phenotype of luminal cells or so-called "breast cancer precursor cells." This cell line retains the phenotype of primary cells as demonstrated by the expression of keratins 18 and 19 and of polymorphic epithelial mucins. However, the cells do not grow in agar after more than 80 passages, nor do they form tumors in nude mice. Established cells contain 2 copies of SV40 DNA integrated into the cellular genome and up to 14 copies of free SV40 DNA. A deletion of the short arm of chromosome 11 (11p15) including the c-Ha-ras and the beta-globin genes was found in the immortalized cells when the DNA from these cells was compared to the DNA from peripheral blood mononuclear cells obtained from the same donor. In addition, this cell line showed a good transfection efficiency for other DNA sequences using classical transfection and selection techniques with a neomycin resistance gene (pKOneo). Selective microinjection of DNA into tumor precursor cells may prove useful for the study of the molecular mechanisms involved in breast carcinogenesis. The possible significance of the loss of 11p13-15 in malignant progression of breast cancer is discussed.

Aneuploidy↗

PubMed1990

Anxiolytic effect of kynurenic acid microinjected into the dorsal periaqueductal gray matter of rats placed in the elevated plus-maze test.

The effect of kynurenic acid (20 to 160 nmol) microinjected into the dorsal periaqueductal gray matter was measured in rats placed in an elevated plus-maze. Microinjection of 160 nmol of kynurenic acid increased the percentages of open arm entries and of time spent in the open arms. Both of these measures may be considered indexes of anxiolysis. Although kynurenic acid also increased the total number of entries, analysis of covariance shows that the increase in open arm entries is independent of the effect on closed arm entries. Thus, the anxiolytic effect of kynurenic acid detected in the elevated plus-maze strengthens the proposal that glutamatergic neurons of the dorsal periaqueductal gray matter play an important role in anxiety.

Analysis of Variance↗

PubMed1985

Intraseptal microinjections of substance P and analogs potentiate pentobarbital-induced narcosis and depression of hippocampal cholinergic activity.

Intraseptal microinjection of Substance P (SP) has been shown to depress activity in the septal-hippocampal cholinergic pathway in the rat. Pentobarbital also depresses septal-hippocampal cholinergic activity, and a relationship between this depressed activity and pentobarbital-induced narcosis is suggested by a variety of studies. To examine this relationship further, we microinjected SP and its analogs, [D-Arg1, D-Pro2, D-Trp7,9, Leu11]-SP and [D-Pro2, D-Trp7,9]-SP, intraseptally in rats pretreated with pentobarbital, and measured the duration of loss of righting reflex and change in choline uptake in hippocampal synaptosomes. The duration of pentobarbital-induced loss of righting reflex was prolonged and the pentobarbital-induced reduction of hippocampal choline uptake was enhanced by all three drugs. A negative correlation (r = -0.96, P less than .02) was seen between duration of loss of righting reflex and synaptosomal choline uptake. Thus, the two analogs of SP appear to act as SP agonists at the septum, judged by their abilities to potentiate pentobarbital narcosis and reduce septal-hippocampal cholinergic activity. This is in contrast to reported actions of these two analogs as SP antagonists in various peripheral tissues.

Animals↗

PubMed1989

Increased susceptibility of detelencephalated rats to audiogenic seizures induced by microinjection of bicuculline into the inferior colliculus.

1. The participation of telencephalic forebrain structures in the induction of audiogenic seizure (AGS) susceptibility and in the behavioral expression of AGS was investigated in rats. 2. Rats that were initially susceptible (N = 12) or non-susceptible (N = 28) to audiogenic seizure were surgically detelencephalated. 3. A unilateral microinjection of a low dose (30 pmol) of the GABA antagonist bicuculline methiodide (BM) was applied to the inferior colliculus (IC) before the animals were exposed to a 120-dB acoustic stimulus. 4. All susceptible rats still exhibited all components of audiogenic seizure after removal of the telencephalon. 5. After BM microinjection, a higher incidence (66% vs 41%) and shorter latencies (6-20 s vs 9-55 s) of occurrence of tonic seizures were observed in the detelencephalated non-susceptible rats when compared to non-operated non-susceptible rats (N = 12). 6. These results suggest that the induction of the behavioural expression of audiogenic seizures is subserved by brain stem neuronal networks but does not require the telencephalon and that telencephalic structures may exert control over audiogenic seizures by inhibiting IC cells through GABAergic neurons.

Acoustic Stimulation↗

PubMed1986

Relationship between anaphase aberrations and carcinogenicity in the trout embryo microinjection assay.

An increased incidence of anaphase aberrations was observed in the tissues of rainbow trout embryos (Salmo gairdneri) within 96 hours of exposure to mitomycin [(MM) CAS: 50-07-7; 50 and 100 ng/embryo] and aflatoxin B1 [(AFB1) CAS: 1162-65-8; 13 and 25 ng/embryo] with the use of the trout embryo microinjection assay. High numbers of anaphase aberrations in embryos exposed to MM were associated with low mitotic indices, cell pyknosis, and high embryo mortalities. When the anaphase aberration data for embryos exposed to AFB1 were compared to previously reported carcinogenesis data from the microinjection assay, the incidence of anaphase aberrations in embryos did not show the same quantitative variations as the incidence of hepatic carcinomas in adult trout. While anaphase aberrations may be an indicator of the clastogenic effects of chemicals on fish embryos, it is unlikely that these aberrations can be used as an early indicator of a carcinogenic response in the trout embryo assay.

Aflatoxin B1↗

PubMed1988

Microinjection into the lateral ventricle: validation of the procedure in estrogen-treated ovariectomized cynomolgus monkey.

To demonstrate that microinjection into the lateral ventricle intracerebroventricular (i.c.v.) of the brain of monkeys does not perturb normal function of the hypothalamic-pituitary axis, ovariectomized cynomolgus monkeys were treated with a bolus of estradiol benzoate (E2beta) (To hours) and an i.c.v. injection of 100 microliters of saline solution at T-6 (group I: four monkeys) or at T+26 hours (group II: three monkeys). The lack of disturbance of the normal biphasic luteinizing hormone response to estrogen and low prolactin (PRL) concentrations allow us to conclude that this microinjection procedure did not disrupt normal endocrine events.

Animals↗

PubMed1985

Quantitative localization of polystyrene microspheres following microinjection in the avian metencephalic neural crest pathway.

Polystyrene microspheres were microinjected into crest populations at two preotic sites in Hamburger-Hamilton stage 10 and 11 chick embryos to investigate factors modulating cephalic neural crest fate. Analyses of microsphere localization and comparisons with cephalic crest fate maps indicated the following: microspheres injected at stage 10 localized with derivatives reflecting the fate of the crest population at the injection site; microspheres injected at stage 11 exhibited minimal displacement; and localization in ectoderm was similar in embryos injected at either stage. These results suggest that microinjected microspheres can be used to investigate normal and abnormal craniofacial morphogenesis.

Animals↗

PubMedDate not supplied

Some behavioral effects of microinjections of serotonin and noradrenaline into the limbic structures of the rat brain.

The effects of microinjections of serotonin (5HT) and noradrenaline (NA) into the limbic structures of the rat brain on the open field behavior and acquisition and retention of a step-down passive avoidance, were examined. Bilateral injections of NA and 5HT into the dorsal hippocampus (DH) produced opposite effects in the open field test, of excitatory and inhibitor nature, respectively. Intrahippocampal injections of 5HT slightly attenuated passive avoidance acquisition and significantly impaired its retention. Bilateral microinjections of NA and 5HT into the basomedial amygdala (BMA) inhibited locomotor activity and exploration in the open field test. In a dose of 40 micrograms, NA and 5HT significantly attenuated also the performance of an avoidance retention, examined 24 h after training. Bilateral injection of NA and 5HT into nucleus accumbens septi (NAS) inhibited 5 min later the locomotor activity and exploration in the open field test. The monoamines administered in various doses into the NAS before training did not consistently influence the rate of acquisition and retention of a passive avoidance reaction. The data are discussed in terms of an involvement of monoaminergic innervation of limbic structures of the brain in the organization of animal behavior.

Amygdala↗

PubMed1981

Serotoninergic control of punished behavior: effects of intra-raphe microinjections of chlordiazepoxide, GABA and 5-HT on behavioral suppression in rats.

The effects of intra dorsalis raphe microinjections of chlordiazepoxide, GABA and 5-HT were studied on a model of behavioral suppression in rats. The suppression of responding for food was elicited by a 10 min presentation of a signal previously associated with the delivery of electric foot shocks. Microinjections of chlordiazepoxide, GABA and 5-HT (0.2 microliters) performed in awake hand-held rats reduced the behavioral suppression. Chlordiazepoxide and 5-HT acted in a synergistic way, while such a synergy could not be obtained when chlordiazepoxide and GABA were coadministered. Moreover, the effects of intra-raphe chloridazepoxide no longer appeared in rats given intra dorsalis raphe 5-7 dihydroxytryptamine (3 micrograms in 0.4 microliters) 3 weeks before testing. All these results further support the hypothesis of the involvement of 5-HT neurons both in punishment-induced inhibition and in the antipunishment activity of benzodiazepines. No evidence was obtained suggesting a GABAergic mediation for the functional interaction of chlordiazepoxide with 5-HT neurons in this experimental model. Since high 3H-flunitrazepam binding was found at the raphe dorsalis level, and since a 30% decrease in the number of intra-raphe 3H-flunitrazepam binding sites was detected after intra-raphe 5-7 dihydroxytryptamine, it is possible that intra-raphe chlordiazepoxide controls 5-HT neurons partly through some benzodiazepine binding sites located on 5-HT cells.

5,7-Dihydroxytryptamine↗

PubMed1996

Microinjection of intact MAP-4 and fragments induces changes of the cytoskeleton in PtK2 cells.

The molecular cloning and sequencing of microtubule-associated protein (MAP)-4 identified microtubule-binding repeats near the C-terminus and a projection domain near the N-terminus. Although it is well known that MAP-4 stimulates the assembly of and stabilizes microtubules (MT) in vitro, the function of MAP-4 in vivo is still unclear. In this study, we examined the function of MAP-4 in the cytoskeleton both in vitro and in vivo. Intact MAP-4 was prepared from bovine adrenal cortex, and the truncated fragments of the N- and the C-terminal halves (named NR and PA4 fragments, respectively) were expressed in Escherichia coli and isolated. In vitro studies demonstrated that in a solution containing a physiological concentration of NaCl, intact MAP-4 and the PA4 fragment were bound to MT, but not to F-actin. The NR fragment was not bound to MT or to F-actin. We also examined the MT changes in PtK2 cells after they had been microinjected with intact MAP-4 and the truncated fragments of PA4 and NR. The injection of intact MAP-4 or PA4 into the cells induced an increase in the number of cytoplasmic MT, as well as MT bundling. The NR fragment did not affect the MT array. Injected MAP-4 and PA4 were associated with the increased MT. In addition, injection with MAP-4 and PA4 stabilized MT in relation to treatment with the MT-disrupting drug, nocodazole. These results indicated that intact MAP-4 and the PA4 fragment promoted MT assembly and stabilized MT, by binding to MT, in vivo as well as in vitro. Further, the injection of the PA4 fragment induced an increase in stress fibers. However, these proteins did not show any association with the stress fibers. Our results suggest that there is an indirect effect of MAP-4 on stress fibers rather than a direct interaction between MAP-4 and stress fibers.

Actins↗

PubMed1997

Monoclonal antibodies recognizing the N- and C-terminal regions of talin disrupt actin stress fibers when microinjected into human fibroblasts.

We have characterized a panel of 6 monoclonal antibodies raised against human platelet talin by Western blotting, immune precipitation, and immunofluorescence, and shown that antibodies TA205 and TD77 disrupt actin stress fibers and focal adhesions, and inhibit cell motility when microinjected into human fibroblasts. Using a series of chick talin fusion proteins spanning the entire length of the molecule, we have mapped the epitopes recognized by these antibodies to the conserved N- and C-terminal regions of the protein. TA205 bound to an epitope contained within residues 139-433, a region which overlaps an F-actin binding site, and which shows homology with the ezrin/radixin/moesin family of cytoskeletal proteins. The epitope recognized by TD77 was located within the C-terminal region of the protein (residues 2269-2541) which also contains an F-actin binding site homologous to that in the yeast actin-binding protein SIa2p. To investigate the possibility that TD77 disrupts actin stress fibers by binding directly to the C-terminal actin binding site, additional talin fusion proteins were generated and analyzed for TD77 and actin binding. Fusion proteins containing residues 2269-2541, 2304-2541, and 2304-2463 all cosedimented with F-actin, whereas TD77 did not recognize the latter fusion protein. These results show that the C-terminal actin-binding site is distinct from the region recognized by the anti-functional antibody TD77, raising the possibility that it binds to a novel functionally important ligand-binding site in the talin molecule.

Actins↗

PubMed1996

In vivo induction of inducible nitric oxide synthase by microinjection with interferon-gamma and lipopolysaccharide in rat hippocampus.

To clarify whether the inducible nitric oxide synthase (iNOS) protein can be induced in in vivo brain, we examined the influence of direct intrahippocampal injection with interferon-gamma (IFN-gamma) plus lipopolysaccharide (LPS) in the rat. In the area surrounding the microinjection site, NOS activity (NO2- accumulation) was enhanced 24 h after injection with IFN-gamma plus LPS. Although the level of 160-kDa nNOS protein was not changed, the 130-kDa iNOS protein was induced 12 h after the injection. On the other hand, iNOS mRNA could be detected at 6 and 12 h but not at 24 h. iNOS immunoreactivity was observed in CD11b-immunopositive microglia in close proximity to the injection site, but the immunoreactivity was not colocalized with glial fibrillary acidic protein-immunopositive astrocytes. Although CD11b-immunopositive microglia were of the ramified type even after injection with vehicle after 24 h, injection with IFN-gamma plus LPS caused numerous microglia to change to the ameboid type and to express major histocompatibility complex (MHC) class II antigens. In some of these ameboidal microglia, iNOS immunoreactivity was observed. These results suggest that intrahippocampal injection with IFN-gamma plus LPS induced iNOS mRNA after 6 h and iNOS protein after 12 h in some of the ameboidal microglia that expressed MHC class II antigens in in vivo rat brain.

Animals↗

PubMed1996

Low levels of chimerism in rabbit fetuses produced from preimplantation embryos microinjected with fetal gonadal cells.

The potential pluripotency of rabbit fetal germ cells has been investigated by using them to make chimeric embryos. Gonial cells, isolated from enzyme-dispersed male and female transgenic fetal rabbit gonads of 18-22 days gestation, were microinjected in groups of about 10 into 640 nontransgenic rabbit embryos between the two-cell and expanded blastocyst stages. Injections were made with primary isolations of gonial cells, within 48 hr of their collection. The injected embryos were transferred, with or without non-injected control embryos, into 49 recipient rabbits. Tissues from 159 resulting fetuses, implantation sites, and a few liveborn young were examined by PCR analysis for the two transgenes used (alpha-1 antitrypsin or luciferase). The overall pregnancy rate (about 80%) was not affected by the stage of development of the embryo injected, nor by co-transfer of control embryos. The survival rate of injected embryos (18% overall, 23.6% in pregnant recipients) was almost identical to that of 243 control embryos. Chimerism was detectable in tissues produced from 4 of 159 (2.5%) of the injected embryos, all four of which had been injected at the 8- to 16-cell stage. This low rate of success indicates that, although passage of rabbit gonial cells is not an absolute requirement for pluripotency, further investigation should pay particular attention to improving culture conditions with a view to deriving EG cell lines.

Animals↗

PubMed1996

Somatic histone H1 microinjected into fertilized mouse eggs is transported into the pronuclei but does not disrupt subsequent preimplantation development.

We injected somatic subtypes of histone H1 into newly fertilized mouse eggs, which do not naturally contain this chromosomal protein, and examined the fate of the injected protein and its effect on preimplantation development of recipient eggs. Rhodamine-labelled H1 injected into the cytoplasm of 53 eggs was transported into the pronuclei in 51 cases, and this nuclear accumulation could be detected within 15 min of injection. Unlabelled histone H1, which was detected using immunofluorescence, was also transported following microinjection to the pronuclei, where it colocalized with the chromatin and remained associated with the nuclei following cleavage to the two-cell stage. Nuclear accumulation of injected H1 was inhibited when injected eggs were incubated in the presence of drugs that prevent mitochondrial electron transport or glycolysis, which indicates that nuclear transport occurs through an energy-dependent process, as previously observed in tissue culture cells. To determine whether the presence of somatic H1 in early embryonic nuclei would influence subsequent development, fertilized eggs were injected with an approximately physiological quantity (1-5 pg) of somatic H1 or, as controls, with another small basic protein, cytochrome c. Fifty-three eggs were injected with cytochrome c, of which 51 divided to the two-cell stage, and 32 (60%) reached the blastocyst stage, after 5 days in culture. One hundred and eleven eggs were injected with somatic H1, of which 95 divided to the two-cell stage, and 53 (48%) reached the blastocyst stage, after 5 days in culture. The two groups did not differ statistically (chi 2, P > 0.1) with respect to the fraction of injected embryos that developed to the blastocyst stage. These results show that, although mouse embryos lack the somatic subtypes of histone H1 until the four-cell stage of development, they are able to progress through preimplantation development when these subtypes are present beginning at the one-cell stage. This may imply that the distinctive chromatin composition that characterizes early embryos of a variety of species is not essential for early development in mammals.

Animals↗

PubMed2000

Characterization of Cre-mediated cassette exchange after plasmid microinjection in fertilized mouse oocytes.

DNA cassette exchange methodologies are powerful tools for rapid modification of defined genomic loci. Until now, most studies have focused on transfected cell lines that were subjected to positive and negative selection to enrich for correctly recombined clones. This is the first report on Cre-mediated cassette exchange (CMCE) in early mouse embryos, a nonselectable biological system. A Cre expression plasmid and a replacement plasmid were introduced into fertilized oocytes by plasmid microinjection and recombination products were investigated. Both, replacement plasmid and oocyte genome carried heterospecific lox sites (lox511 and loxP) to direct CMCE toward integration. Here, we demonstrate the general feasibility of CMCE in early mouse embryos and characterize the system with respect to integration efficiency, competing reactions and parameters affecting recombination rates.

Animals↗

PubMed1999

Tremor arrest with thalamic microinjections of muscimol in patients with essential tremor.

Six patients undergoing stereotactic procedures for essential tremor received microinjections of muscimol (a gamma-aminobutyric acid-A [GABA(A)] agonist) into the ventralis intermedius thalamus in areas where tremor-synchronous cells were identified electrophysiologically with microelectrode recordings and where tremor reduction occurred with electrical microstimulation. Injections of muscimol but not saline consistently reduced tremor in each patient. The effect had a mean latency of 7 minutes and lasted an average of 9 minutes. We propose that GABA-mediated thalamic neuronal inhibition may represent a mechanism underlying the effectiveness of surgery for tremor and that GABA analogues could potentially be used therapeutically.

Aged↗

PubMed1982

Effects of phalloidin microinjection and localization of fluorescein-labeled phalloidin in living sand dollar eggs.

Effects of microinjection of phalloidin on fertilization and cleavage of sand dollar (Clypeaster japonicus and Scaphechinus mirabilis) eggs were studied. The drug, previously injected into unfertilized eggs, showed no effect on the elevation of the fertilization membrane upon insemination up to an intracellular concentration of 50 microM. However, the movement of the egg pronucleus to the sperm pronucleus was inhibited and the fusion of pronuclei did not occur. The subsequent development no longer took place. When phalloidin was injected into fertilized eggs, the thickness of the cortical layer increased and the microvilli became conspicuous. Both nuclear division and cleavage were inhibited at the intracellular concentration of more than 20 microM, though the latter seemed to be more sensitive to phalloidin than the former. Fluorescein-labeled phalloidin (FL-phalloidin) was injected into eggs in order to investigate F-actin localization by fluorescence microscopy. In both unfertilized and fertilized eggs, FL-phalloidin was localized in the cortical layer within 1 min after injection. It was also localized in the cortical layer as radially oriented rod-like structures when injected into fertilized eggs before the disappearance of the nuclear membrane. No distinct fluorescence was detected in the mitotic apparatus or in the cleavage furrow. FL-phalloidin redistributed gradually into egg cytoplasm. In unfertilized eggs, fluorescent rods were found especially in the egg pronucleus 30 min after injection.

Actins↗