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PubMed2000

Temporal, regional, and cell-specific changes of iNOS expression after intrastriatal microinjection of interferon gamma and bacterial lipopolysaccharide.

Here we study expression of the inducible isoform of nitric oxide synthases after intrastriatal microinjection of interferon-gamma and bacterial lipopolysaccharide in the rat at different time points to detect time- and localisation-dependent changes of iNOS expression. Three different areas in the striatum and the corpus callosum were evaluated. Antibodies against the glial fibrillary acidic protein and the microglia/brain macrophage epitope ED1 were used to detect colocalization of inducible nitric oxide synthase with astrocytes or activated microglia/brain macrophages, respectively. Inducible nitric oxide synthase-positive cells occurred first in intravascular and perivascular cells at 4 h. Perivascular and parenchymal inducible nitric oxide synthase expression increased up to 24 h in the striatum, whereas in the corpus callosum inducible nitric oxide synthase expression was maximal after 16 h. Inducible nitric oxide synthase was still present in perivascular cells 7 days after immunostimulation. At all time points, inducible nitric oxide synthase was predominantly detected in ED1-positive microglia/brain. Nitrotyrosine immunohistochemistry was performed to detect NO-mediated nitration of proteins at all time points. Nitrotyrosine-positive neurons and microglial cells were detected from 24 h until 7 days after immunostimulation and were absent in controls. Detailed knowledge of the changes in the time course and cellular source of inducible nitric oxide synthase expression following brain immunostimulation provide a basis for establishing treatment strategies and windows of therapeutic intervention during neuroinflammation.

Animals↗

PubMed2001

Differential effects of NMDA antagonists microinjections into the nucleus reticularis pontis caudalis on seizures induced by pentylenetetrazol in the rat.

It has been shown that NMDA antagonists block the tonic but not the clonic component of seizures when they are injected in the oral region of the rat pontine reticular formation (PRF). The participation of the caudal PRF in the effects of NMDA antagonists upon the tonic and the clonic components of generalized seizures induced by pentylenetetrazol (PTZ) is unknown. The aim of the present study was to evaluate the effects of unilateral microinjections of competitive and non-competitive NMDA antagonists, 2-amino-7-phosphonoheptanoic acid (AP-7) and dizocilpine (MK-801), respectively, into the nucleus reticularis pontis caudalis of the rat PRF upon seizures induced by PTZ (70 mg/kg i.p.). MK-801 induced a dose-related decrease both in the incidence of generalized tonic-clonic seizures (GTCS) and in the presence of spikes in the EEG. MK-801 also increased GTCS latency. On the contrary, AP-7 did not have effects on GTCS. Interestingly, it induced ipsilateral circling behavior. These results suggest that in the caudal region of the rat PRF only non-competitive NMDA antagonists should block the generation of tonic and clonic components of generalized seizures.

2-Amino-5-phosphonovalerate↗

PubMed2000

Seizures induced by penicillin microinjections in the mesencephalic tegmentum.

The location and extension of a convulsive area in the brain stem in cats was determined through penicillin microinjections (0.5-1.0 microl) of a concentrated sodium penicillin solution (500 IU/microl), stereotactically oriented to multiple structures, in fully awake animals, partially restrained through a rod fixation system that avoided pain, allowed the observation of clinical seizures and simultaneous recording of EEG, EMG and multiple unit activity (MUA) from the injected site and the motor cortex (Cx). Clinical and EEG seizure patterns in relation to the injected sites and penicillin doses were studied in another group of animals using doses from 12.5 IU/0.1 microl to 125 IU/1.0 microl. The time relationship between muscular clonus, EEG spikes and MUA at the injected site and Cx were analyzed. The only area in which penicillin induced seizures was the mesencephalic tegmentum (MT). The amount of penicillin but not the stereotactic coordinates determined the seizure type. MT EEG and MUA paroxysms anticipated clinical seizure and Cx EEG spikes. When Cx EEG appeared, they were accompanied by an increase in MUA beginning in the Cx and EMG, followed by significant increase in MT MUA. The sequence of events suggest that MT seizure activity propagates via alternative pathways not involving direct reticulospinal or pyramidal tract pathways.

Animals↗

PubMed2002

Effects of 5-HT(1B) receptor ligands microinjected into the accumbal shell or core on the sensitization to cocaine in rats.

The present study was designed to find out whether 5-HT(1B) receptors located in subareas of the nucleus accumbens played a role in cocaine sensitization in rats, and whether pharmacological activation of these receptors could modify this drug effect. Male Wistar rats implanted bilaterally with cannulae into the accumbens shell or core were microinjected with GR 55562 (an antagonist of 5-HT(1B) receptors) or CP 93129 (an agonist of 5-HT(1B) receptors). The rats, which were repeatedly (for 5 days) administered with cocaine (10 mg/kg) and then challenged with cocaine (10 mg/kg) after 5-day withdrawal period, showed significantly higher locomotor hyperactivity in comparison with the effect observed in saline-pretreated and cocaine challenged rats. GR 55562 (0.1-10 microg/side), administered for 5 days into the accumbens shell, but not into the core, prior to cocaine dose-dependently attenuated cocaine sensitization. When injected for 5 days into either the accumbens shell or core before cocaine, CP 93129 (0.1-10 microg/side) had no effect on the development of cocaine sensitization. To examine the effects of GR 55562 and CP 93129 on the expression of cocaine sensitization, the drugs were given acutely before a challenge dose of cocaine (10 mg/kg) on day 10. No change in cocaine sensitization was observed after injection of GR 55562 (0.1-10 microg/side) to the either accumbens subregion or after injection of CP 93129 (0.1-10 microg/side) into the core. However, an intra-accumbens shell injection of CP 93129 (10-30 microg/side) increased sensitization to cocaine, and this enhancement was attenuated after local injection of GR 55562 (1 microg/side). Our findings indicate that behavioral sensitization to cocaine may be modulated by 5-HT(1B) receptor ligands in the accumbens shell, but not into the core. They suggest that the inhibition of 5-HT(1B) receptors in the accumbens shell attenuates the development, whereas their pharmacological activation enhances the expression of cocaine sensitization.

Analysis of Variance↗

PubMed1994

Microinjected profilin affects cytoplasmic streaming in plant cells by rapidly depolymerizing actin microfilaments.

BACKGROUND: Cytoplasmic streaming is a conspicuous feature of plant cell behaviour, in which organelles and vesicles shuttle along cytoplasmic strands that contain actin filaments. The mechanisms that regulate streaming and the formation of actin filament networks are largely unknown, but in all likelihood involve actin-binding proteins. The monomeric actin-binding protein, profilin, is a key regulator of actin-filament dynamics in animal cells and it has recently been identified in plants as a pollen allergen. We set out to determine whether plant profilin can act as a monomeric actin-binding protein and influence actin dynamics in plant cells in vivo. RESULTS: Recombinant birch-pollen profilin was purified by polyproline affinity chromatography and microinjected into Tradescantia blossfeldiana stamen hair cells. After profilin injection, a rapid and irreversible change in cellular organization and streaming was observed: within 1-3 minutes the transvacuolar cytoplasmic strands became thinner and snapped, and cytoplasmic streaming ceased. Fluorescein-labelled-phalloidin staining confirmed that this was due to depolymerization of actin filaments. To confirm that the effects observed were due to sequestration of monomeric actin, another monomeric actin-binding protein, DNase I, was injected and found to produce comparable results. CONCLUSIONS: Profilin can act as a potent regulator of actin organization in living plant cells. Its rapid effect on the integrity of cytoplasmic strands and cytoplasmic streaming supports a model in which organelle movements depend upon microfilaments that exist in dynamic equilibrium with the pool of monomeric actin.

Actin Cytoskeleton↗

PubMed1996

Microinjection into giant vesicles and light microscopy investigation of enzyme-mediated vesicle transformations.

BACKGROUND: 'Giant vesicles' have diameters of several micrometers and can be observed by light microscopy. Their size may allow manipulation of individual vesicles and direct observation of the progress of a chemical reaction in real time. We set out to test this possibility using enzymatic hydrolysis of vesicle components as a model system. RESULTS: We describe a novel micromanipulation technique that allows us to microinject femtoliter amounts of a reagent solution adjacent to or into giant vesicles with diameters ranging from 10 to 60 microm. The vesicle transformations can be monitored directly in real time by light microscopy and recorded by video analysis. Snake venom phospholipase A2 was added to vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine, and the enzymatic hydrolysis of components of the lipid bilayer was observed over time. A specific effect on the targeted giant vesicle was seen and video recorded, while the neighbouring vesicles remained unaffected. Addition of the enzyme to the outside of a vesicle caused it to burst, whereas injection of the enzyme inside a vesicle resulted in a slow and constant decrease in its size, until it eventually disappeared from the resolution power of the light microscope. CONCLUSIONS: These results show that it is possible to micromanipulate an individual vesicle, and to follow visually the progress of an enzymatic reaction occurring on the vesicle bilayer over time.

Microinjections↗

PubMed1999

A simple pressure microinjecting system for delivery of small substance volumes to the brain: application to the developmental study of thalamo-cortical projections in foetal and neonatal rats.

We describe a reliable and inexpensive method for placing injections of anatomical tracers into the brain of lower mammals. The pressure microinjecting system we developed is specifically designed to deliver very small amount of substances. The injecting portion of the system is relatively easy to assemble and can be repeatedly used for multiple experimental sessions. The system has been validated with experiments of multiple fluorescent retrograde tracing. In these experiments the populations of thalamo-cortical neurons were consistently labeled by the tracers injected bilaterally and symmetrically in the cortex of foetal and neonatal rats.

Amidines↗

PubMed2003

Effects of vasoactive intestinal polypeptide microinjected into the nucleus tractus solitarius on jejunal glucose absorption in rats.

It has been shown that vasoactive intestinal polypeptide (VIP) injected into the nucleus tractus solitarius and into the dorsal motor nucleus of the vagus inhibits alanine absorption across the jejunum. The aim of the present study was to investigate the effects of VIP injection into the nucleus tractus solitarius on jejunal absorption of glucose in the rat. Forty Wistar rats were submitted to midline laparotomy to expose and isolate 20 cm of jejunal loop and to perform a subdiaphragmatic troncular vagotomy. Saline or VIP (10 pg 100 nl(-1)) was injected into the rostral nucleus tractus solitarius using a stereotaxic instrument. Tyrode solution, pH 8, containing twice glucose, sodium, and potassium concentrations was infused (0.5 ml min(-1)) into the jejunal loop. Samples were taken at 10-min intervals during the 40-min experiment. Injection of VIP into the nucleus tractus solitarius associated with vagotomy resulted in inhibition of jejunal glucose absorption by VIP alone at 10 and 40 min after perfusion (2.75+/-0.19 vs. 3.53+/-0.29 mg). The vagal outflow tract maintained jejunal glucose absorption even when VIP was microinjected into the nucleus tractus solitarius.

Animals↗

PubMed1993

Microinjection of FITC-dextran into mouse blastomeres to assess topical effects of zona photoablation.

The objective of the current experiments was to investigate whether all or only some blastomeres from precompacted mouse embryos were affected by zona photoablation. The microbeam of xenon chloride excimer laser (308 nm) was guided through an inverted microscope (non-contact system). Topical effects of lasing were determined by microinjection of a vital fluorescent dye of high molecular weight (fluorescein isothiocyanate [FITC] dextran) into the cell immediately adjacent to the site of zona photoablation. This dye is only passed onto daughter blastomeres and therefore allows study of specific cell lines. Embryonic growth was assessed following cell separation at the morula and blastocyst stage. Four-cell embryos treated with the laser had significantly fewer cells 12 h after zona photoablation than control embryos. A similar effect was noted after 24 h between dye injected embryos and those injected and lased simultaneously, indicating potential toxic effects of the laser treatment on the embryo. Effects on the blastomere closest to the site of ablation were evaluated by calculating the ratio of dyed cells to the total number of cells at specific time intervals. The ratios were similar in the dye and laser+dye groups of treated 4-cell embryos 36 h after treatment (0.22 and 0.23, respectively), indicating that the dye was still present in approximately 25% of the cells and that the negative effect of photoablation was evenly distributed among the blastomeres. It is concluded that zona photoablation may have long-term detrimental effects of a non-topical nature on precompacted mouse embryos in spite of the apparent precision of the laser spot size.

Animals↗

PubMed1994

Isolation and microinjection of active sperm nuclei into egg cells and central cells of isolated maize embryo sacs.

Artificial fertilisation was attempted in maize by microinjecting sperm nuclei into the egg cell or central cell of isolated embryo sacs. A protocol for isolation of nuclei from pollen grains was developed and a pure fraction of sperm nuclei was obtained after centrifugation on a Percoll gradient. The in vitro transcriptional activity of the nuclei was tested by incorporation of radioactive UTP into RNA. The level of labelled nucleotide incorporation increased and reached a maximum after between 30 and 40 min in the incubation medium. The embryo sacs were enzymatically isolated and their viability determined by observation of cytoplasmic streaming in the female cells. The embryo sacs were immobilised by embedding in low-melting-point agarose and a single male nucleus was injected with a bevelled microcapillary. The presence of the injected nucleus in the egg or central cell was demonstrated using a cytological approach. This paper presents an alternative method for studying the intimate processes of fertilisation in plants.

Fertilization↗

PubMed1995

Early mouse preimplantation development is unaffected by microinjection of metallothionein antibodies.

Polyclonal antibodies that cross-react with rodent metallothionein I (MT I) and metallothionein II (MT II) were microinjected in 1-cell and 2-cell mouse embryos, into either the cytoplasm or the nucleus. Regardless of the experimental treatment, mouse embryo development in vitro was not affected and most of the embryos cleaved normally until the morula stage. The results suggest that metallothionein is not essential for normal mouse early preimplantational development, in agreement with recent studies in mice with inactivated MT I and MT II genes.

Animals↗

PubMed2002

DNA replication and RNA synthesis in thymocyte nuclei microinjected into the cytoplasm of artificially activated mouse eggs.

Thymocyte nuclei were microinjected into the cytoplasm of parthenogenetic mouse eggs within 60 min or 3 h after egg activation and DNA replication and RNA synthesis were analysed in remodelled thymocyte nuclei and female pronuclei. We show that thymocyte nuclei which transform into pronucleus-like nuclei (thymocytes injected not later than 60 min after activation) enter S-phase 1 h earlier than the female pronuclei. At the beginning of the first cell cycle they remain transcriptionally silent, but in G2 undertake transcription earlier than the female pronuclei. Partly remodelled thymocyte nuclei (injected 3 h after activation) start to replicate DNA at the same time as the female pronuclei. They reinitiate RNA synthesis within 2 h after transfer and continue to transcribe irrespective of the transcriptional activity of the female pronucleus. We show that the observed transcription is only nuclear, i.e. RNA polymerase II-dependent.

Animals↗

PubMed2005

Survival and ultrastructure of gene-microinjected rabbit embryos after vitrification.

Morphological signs of injury and subsequent regeneration following vitrification of either rabbit gene microinjected (Gene-Mi) or intact in vitro cultured embryos derived from in vivo fertilized eggs were evaluated by post-warming recovery in culture and analysed by transmission electron microscopy (TEM). The percentages of vitrified/warmed Gene-Mi embryos that reached the blastocyst stage (69%) and hatched (57%) did not differ significantly from those of intact embryos (78% and 56%, respectively). In contrast, in vitro development of embryos to the blastocyst stage among non-vitrified intact (96%) and Gene-Mi (90%) embryos compared with both the intact vitrified (78%) and Gene-Mi vitrified (69%) groups, as well as hatching rate (94%, 90% vs 56%, 57%, respectively) varied significantly (p < 0.001). Observations by TEM showed that the vitrified/warmed intact or Gene-Mi embryos without post-culture displayed severe degenerative changes among their cells. During 24 h of culture a proportion of the embryos were able to regenerate and complete the compaction process. Nevertheless the signs of previous injury were retained, such as swollen cytoplasmic organelles and remaining cellular debris in the perivitelline space. These observations indicate that the procedure of gene Mi does not significantly compromise embryo tolerance to cryopreservation and post-warming developmental ability. Severe changes in embryo morphology, observed at the ultrastructural level, can be attributed to a direct influence of the vitrification process rather than to the Mi procedure itself.

Animals↗

PubMed1998

Fate of microinjected spermatid mitochondria in the mouse oocyte and embryo.

Mouse round spermatids labelled with MitoTracker were microinjected into Sr(2+)-activated mouse oocytes. The labelled mitochondria were tracked up to the morula/blastocyst stage using fluorescence microscopy. The overall incidence of embryos with labelled mitochondria fell from 80% in the 1-cell zygote to 25% in 2-cell, 9% in 4-cell and approximately 1% in 8-cell or later stages. Thus it appears that almost all round spermatid mitochondria finally disappear from embryos during the 4-cell to 8-cell transition, as happens for mature spermatozoa (Cummins et al. Zygote 1997, 5:301-8). The spermatid mitochondria remained tightly bound together during this process. In contrast, labelled primary spermatocyte and cumulus mitochondria dispersed rapidly throughout the oocyte cytoplasm within 3 h. We hypothesise that spermatid mitochondria may be bound together by cytoskeletal elements produced in the early haploid spermatid. These elements, together with terminal differentiation of the sperm mitochondria, may be central to the processes by which the embryo 'recognises' the sperm mitochondria and inhibits inheritance of paternal mitochondrial DNA. These results suggest that round spermatid injection for infertile men will not pose a significant risk to offspring by transmitting abnormal mitochondrial genomes.

Animals↗

PubMed1999

Fate of genetically marked mitochondrial DNA from spermatocytes microinjected into mouse zygotes.

Cytoplasts from single spermatocytes of NZB/BinJ mice were separated from the nuclei and individually microinjected into B6D2F1 (C57BL/6 x DNBA/2J) hybrid embryos at the pronuclear stage (20 h after hCG injection). Of 363 zygotes injected, 311 (86%) survived and developed. From these experiments, we transferred 222 embryos into 20 pseudopregnant recipients. Eighteen (90%) became pregnant and 82 pups were born (37% of transfers). Mitochondrial DNA (mt DNA) from the NZB/BinJ strain lacks a RsaI restriction site and can thus be distinguished from the host embryo following PCR amplification. We were unable to detect the transferred mtDNA in blastocysts on day 4-5 after injection. Nor could we detect NZB/BinJ mtDNA in placentae, nor in tissues from mice born to host mothers following the transfer of blastocysts that developed from injected zygotes. Rejection of paternal mitochondria by the embryo normally occurs at the 4- to 8-cell stage in mice and is apparently dependent on mutual recognition between the mitochondria and the nuclear genome. We conclude that this mechanism has probably already developed by the time the germ cells have become committed to meiosis.

Animals↗

PubMed1999

Persistence of human mitochondrial DNA throughout the development to the blastocyst of mouse zygotes microinjected with human mitochondria.

The conditions for transfer of human mitochondria into fertilised mouse ova were elaborated. Species-specific primers were designed to discriminate human mitochondrial DNA (mtDNA) and the endogenous mtDNA in the preimplantation embryos. Human mitochondria isolated from the HepG2 cell line were microinjected into murine zygotes, and the latter cultured for 96 h to the blastocyst stage. The polymerase chain reaction allowed the detection of human mtDNA at every stage of embryo cleavage. In some cases a clear disparity in distribution of human mtDNA among blastomeres was evident.

Animals↗

PubMed1979

In vivo studies on the incorporation of microinjected acidic proteins of the large ribosomal subunit from Escherichia coli and artermia salina into oocyte ribosomes from Xenopus laevis.

Tritium-labeled acidic proteins from the large ribosomal subunit of Artermia salina or Escherichia coli were microinjected into the cytoplasm of stage IV/V oocytes from Xenopus laevis. eL12 from the large ribosomal subunit of A. salina but not L7/L12 or L7/L12--L10 from E. coli is specifically incorporated into 60S ribosomal subunits of oocytes. This incorporation is not significantly inhibited by actinomycin D. Incorporation of eL12 into the 60S subunits occurs in enucleated oocytes, suggesting that active ribosomal ribonucleic acid synthesis and ribosome assembly as well are not prerequired for this reaction. Apparently the incorporation proceeds via an exchange reaction between a free cytoplasmic pool of eL12 and ribosomal eL12.

Animals↗

PubMed1998

RNA expression from a site-specific non-LTR retrotransposon microinjected into Xenopus oocytes.

Tx1L is a site-specific non-LTR retrotransposon (NLR) that has been identified in the genome of Xenopus laevis. Using microinjection into Xenopus oocytes, several aspects of RNA expression by these elements were investigated. With constructs carrying various parts of the element we saw no evidence of promoter activity, unlike what has been shown for several other elements of this class. Tx1L transcription was induced by linking a whole element to a promoter that is active in oocytes. Among the RNAs produced, about half had 3' ends located near the end of the element, suggesting that instruction for 3' end formation are encoded in the element or its target. Deletion of the 3' UTR of Tx1L and of surrounding target sequences indicated that these regions are not required for termination or processing of the RNA. PolyA or very A-rich sequences were added at these 3' ends, despite the absence of canonical polyA addition signals. A significant proportion of non-A residues was found in the 3' untemplated tails, and this is reminiscent of non-templated insertions often found at the 3' junction of new genomic copies of some NLRs.

3' Untranslated Regions↗