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PubMed1989

Microinjection and expression of a mouse metallothionein human growth hormone fusion gene in fertilized salmonid eggs.

Using a microinjection method (Rokkones et al. 1985) deoxyribonucleic acid was introduced into fertilized salmonid eggs. The survival rate after a 28 day period was 91% for injected eggs in comparison to non-injected controls. A gene construct containing the mouse metallothionein promoter fused to the human growth hormone structural gene was microinjected either as a supercoiled plasmid or as a linear sequence. In Southern blot analysis of both 5 and 73 day old dissected rainbow trout embryos, as well as in 1 year old Atlantic salmon, the mouse metallothionein human growth hormone gene sequence was detected together with the chromosomal DNA when micro-injected as plasmid or as linear DNA. After digestion with Bam HI restriction endonuclease, the human growth hormone gene was excised from the high molecular weight DNA fraction. Transcription into human growth hormone specific RNA, as well as translation and release of human growth hormone immunoreactive protein, could be demonstrated in early embryonic stages.

Animals↗

PubMed1991

Transfer of the human HPRT and GART genes from yeast to mammalian cells by microinjection of YAC DNA.

DNA of two yeast artificial chromosomes (YACs) containing selectable human genes was transferred by microinjection to rodent cells in tissue culture. The human hypoxanthine phosphoribosyltransferase (HPRT) gene, spanning 45 kb, is contained on the 660-kb YAC yHPRT as described elsewhere. The human phosphoribosylglycinamide formyltransferase (GART) gene, spanning approximately 40 kb, is contained on the 590-kb YAC yGART2 as described previously. YAC DNA was isolated from pulsed-field gels and microinjected into mammalian cells in which the human HPRT and GART genes can be selected. The cell lines that were selected contain the entire human genes. Some of the cell lines contain multiple copies of the genes integrated at the same chromosomal position. The YAC yGART2 could not be purified away from natural yeast chromosomes of similar size, and the cell lines into which the human GART gene was introduced contain variable amounts of yeast DNA in addition to the human DNA.

Acyltransferases↗

PubMed1993

Effects of hypothalamic microinjection of PGE2 on body temperature and sympathetic nervous activities in the rabbit.

The febrile response and sympathetic nervous response to hypothalamic microinjections of prostaglandin E2 (PGE2) were investigated in anesthetized rabbits. Microinjection of PGE2 (500-1000 ng) caused an increase in rectal temperature of more than 0.3 degrees C in 13 of 50 loci in the preoptic and anterior hypothalamic area (PO/AH). At 8 of these 13 loci, PGE2 elicited response patterns in the sympathetic nervous system, such as an increase in cutaneous sympathetic nervous activity and decrease in renal sympathetic nervous activity. This pattern of sympathetic nervous responses was induced with a simultaneous increase in rectal temperature of more than 0.5 degrees C. The 8 loci were distributed in the preoptic area, especially in the vicinity of the supraoptic nucleus. Electrolytic lesions of this region were made bilaterally, and intracerebroventricular injection of PGE2 (8 micrograms/kg) was found to inhibit fever and sympathetic activity. The results demonstrate that the action of PGE2 is responsible for the response patterns of sympathetic twigs during fever. The preoptic area, especially in the vicinity of the supraoptic nucleus, is most sensitive to PGE2 for the patternized response of sympathetic neurons and fever.

Acclimatization↗

PubMed1995

Human sperm chromosome analysis after microinjection into hamster oocytes.

PURPOSE: Subzonal sperm insemination (SUZI) into hamster oocytes was performed to establish the karyotypes of the fertilizing spermatozoa. METHODS: Spermatozoa from two males with normal semen parameters were microinjected. Of 72 (52 + 20) analyzed sperm chromosome metaphases, only 1 (1.4%) was considered abnormal, showing a structural abnormality. RESULTS: No hyperhaploidy was observed. Rates of sperm chromosomal abnormalities after microinjection were not higher than those reported previously using zona-free egg insemination, suggesting that the SUZI procedure per se does not increase sperm chromosomal abnormalities. CONCLUSIONS: The use of subzonal insemination into hamster oocytes for the study of human sperm chromosomes in males with low sperm counts is discussed.

Animals↗

PubMed1996

Microinjection of in vitro transcribed RNA and antisense oligonucleotides in mouse oocytes and early embryos to study the gain- and loss-of-function of genes.

Mouse oocytes have proven useful in experiments aimed at studying gene function. They have been used to analyze the gain-of-function acquired after microinjection of RNA transcribed in vitro from specific gene constructs, and for establishing loss-of-function mutation obtained by injecting in vitro transcribed antisense RNA and/or synthetic oligonucleotides. This article presents protocols utilized in these studies. Specifically, the acquisition of mouse oocytes and/or embryos, the genesis of the necessary DNA and/or RNA to be used, and procedures for microinjection.

Animals↗

PubMed1997

Aging, hyaluronidase removal of the cumulus, and microinjection do not affect the sperm binding potential of human oocytes.

PURPOSE: We studied the influence of aging, hyaluronidase removal of the cumulus, and microinjection on the sperm binding potential of human oocytes under intact zona assay conditions to determine the safe use of unstored aged unfertilized ICSI oocytes on zona binding tests. Results were also compared with those for aged IVF oocytes under the same conditions. METHODS: To avoid the large variation in sperm binding scores, we compared the ratios of the number of sperm bound to nontreated versus to treated oocytes. Treated oocytes were those whose zonae were exposed to hyaluronidase, an agent that decreases sperm binding. Experimental groups were fresh oocytes, experimentally aged fresh oocytes, fresh oocytes whose cumulus was removed either mechanically or with hyaluronidase, aged ICSI oocytes, and aged IVF oocytes. RESULTS: Statistical analysis within and between groups showed that aging, previous insemination, cumulus removal by hyaluronidase, and microinjection did not affect the sperm binding potential of human oocytes. CONCLUSIONS: As the zona binding ability of fresh and unfertilized aged ICSI oocytes is similar, they can be used safely in zona assays.

Aging↗

PubMed2005

Anxiolytic-like effects induced by nitric oxide synthase inhibitors microinjected into the medial amygdala of rats.

RATIONALE: The amygdaloid complex plays a central role in the neuroanatomical circuits that coordinate defensive responses. Nitric oxide (NO) has been involved in the neurochemical, hormonal, and behavioral changes related to stress and anxiety. A high density of NO-producing neurons is observed in the medial amygdala (MeA). These neurons are activated after exposure to threatening stimuli such as a live predator. OBJECTIVE: To test the hypothesis that microinjection into the MeA of two NO synthase inhibitors, NG-nitro-L-arginine methyl ester (L-NAME) or 7-nitroindazole (7-NI), would produce anxiolytic effects. METHODS: Male Wistar rats (n=8-10/group) were submitted to stereotaxic surgery to implant bilateral stainless steel guide cannulae aimed at the MeA. Six days after the surgery, the animals received intra-MeA microinjections of the drugs or vehicle and, 10 min later, were submitted to the elevated plus-maze (EPM) or the light-dark transition procedures. RESULTS: Both L-NAME (50-200 nmol) and 7-NI (5 and 10 nmol) increased open-arm exploration in the EPM without changing the number of enclosed arm entries, indicating an anxiolytic-like effect. The anxiolytic-like effect of L-NAME (200 nmol) was prevented by pretreatment with L-arginine (100 nmol). Injections of 7-NI or L-NAME outside the MeA did not produce any significant change in EPM exploration. When tested in the light-dark test, L-NAME (200 nmol) or 7-NI (10 nmol) increased the time when the animal remained in the light compartment of the light-dark box. CONCLUSIONS: The present results suggest that inhibition of NO formation in the MeA produces anxiolytic-like effect in rats.

Amygdala↗

PubMed2005

Decreased gap junctional communication in neurobiotin microinjected lens epithelial cells after taxol treatment.

The aim of the study was to examine gap-junction-mediated intercellular communication after experimentally induced aggregations of microtubules in cultured bovine lens epithelial cells. Intercellular communication between lens cells appears to be crucial for normal lens homeostasis. However, investigations on the maintenance of direct ion and metabolite exchange via gap junctions and its quantified dependency of cytoskeletal microtubules have not been available under conditions leading to bundling of microtubules. Thus, metabolic coupling of neighboring lens epithelial cells was quantified following microinjections of neurobiotin into single cells under various conditions. In controls, intensive gap-junction-mediated intercellular communication could be documented by dye-spreading of microinjected neurobiotin. In contrast, taxol treatment for 1-3 days impaired, but did not completely block gap-junction-mediated intercellular communication. After depletion of taxol, a complete recovery of intercellular communication was achieved. In addition, confocal laser scanning microscopy and rapid-freeze deep-etch electron microscopy revealed a displacement of actin-filaments from the perinuclear cytoplasm, accompanied by an abnormal aggregation of microtubules after taxol treatment, including impeded translocation of connexin 43 from the cytoplasm into the plasma membrane. Incubation of cells with nocodazole destroyed the microtubule network, accompanied by a clear reduction of plasma-membrane-integrated connexin 43 and significant impairment of dye spreading. Thus, in lens epithelial cells intercellular communication at gap junctions made by connexin 43 depends on the integrity of the microtubule network through the translocation of connexins to the plasma membrane.

Actin Cytoskeleton↗

PubMed2000

Taxol impairs anterograde axonal transport of microinjected horseradish peroxidase in dorsal root ganglia neurons in vitro.

We have investigated the effects of taxol on the axonal transport of horseradish peroxidase (HRP) in dorsal root ganglia (DRG) cells and their neuronal cytoskeleton. The former were analysed by microinjection of HRP into single DRG cells and the latter was studied by means of immunohistochemistry and cryo-electron microscopy. In cultured and untreated DRG cells, microinjected HRP was typically transported anterogradely several hundred micrometres along their neurites. Different exposure periods (1, 2 and 3 days) to taxol were analysed. The axonal transport of HRP in DRG cells was time-dependently impeded by taxol. After the drug had been washed out, a recovery of the axonal transport of HRP was observed and confirmed by quantitative analysis. Cryo-electron microscopy revealed an abnormal aggregation of axonal and cytoplasmic microtubules, associated with a decreased amount of cross-linking structures, in taxol-treated DRG cell cultures. After 3 days of taxol exposure, microtubule-associated proteins and Tau-protein were restricted to the cellular somata but the neurofilament network and tubulin-proteins seemed to be unaffected. Our results demonstrate, for the first time, an inhibition of anterograde axonal transport of HRP in single neurons by taxol. This effect is reversible and seems not to be caused by cellular damage, but is rather a consequence of an altered organisation of microtubules and/or microtubule-associated proteins.

Animals↗

PubMed2006

Respiratory reactions to microinjection of bombesin into the solitary tract nucleus and their mechanisms.

Acute experiments were performed on urethane-anesthetized adult laboratory rats to investigate the effects of microinjections of 10(-13)-10(-4) M bombesin into the solitary tract nucleus on measures of respiration. Bombesin microinjections were found to stimulate respiration, inducing significant increases in the level of pulmonary ventilation, increases in respiratory volume, and increases in the bioelectrical activity of the inspiratory muscles. The most marked respiratory reactions were seen after intermediate peptide doses (10(-10)-10(-7) M). These respiratory effects of bombesin were found to result from its ability to suppress the inspiration-inhibiting Hering-Breuer reflex at the level of the solitary tract nucleus. The fact that ultralow doses of bombesin were active, along with the distribution of endogenous bombesin and its specific receptors in the solitary tract nucleus, and the ability of this peptide to modulate the Hering-Breuer reflex all provide evidence that bombesin is involved in controlling respiration at the level of the dorsal structures of the respiratory center.

Animals↗

PubMed1988

Identification of microinjected cells using biotinylated antibodies and Strep-avidin-conjugated horseradish peroxidase.

Results from experiments using needle microinjection of cells are often compromised by an inability to readily demonstrate which cells within a population have been injected. The technique described here allows the unambiguous identification of cells that have been successfully microinjected. Sequential incubation of fixed cells with biotinylated anti-immunoglobulin antibodies, followed by horseradish peroxidase (HRP)-conjugated Strep-avidin and HRP substrate, provides a sensitive assay for identification of cells containing trace amounts of immunoglobulins. This allows direct correlation to the presence of injected molecules of effects on cell morphology, the ability to enter into DNA synthesis, or expression of specific genes. By a variety of criteria, nonspecific immunoglobulins do not adversely affect cellular processes when injected by themselves or in the presence of other proteins known to have biological effects when injected, such as cAMP-dependent protein kinase and the ras oncogene protein.

Animals↗

PubMed1985

Microinjection of macromolecules into leukemic cells by cell fusion technique: search for intracellular growth-suppressive factors.

To investigate the intracellular molecular events during leukemic cell proliferation, we have examined the method of ghost-mediated microinjection of macromolecules into leukemic cell line cells (HL-60). Samples were packed into red cell ghosts. Microinjection was performed by the fusion of ghosts and HL-60 cells using the hemagglutinating virus of Japan (HVJ). Fusion rate was about 80-90%, when determined by the injection of FITC-labeled globulins (IgG) or diphtheria toxin fragment A into HL-60 cells. When the nuclear protein extract from normal granulocytes was injected into HL-60 cells, their growth was significantly suppressed. The injection of the nuclear protein extract from HL-60 itself into HL-60 cells did not inhibit their growth. This finding suggests that leukemic cells may be deficient in intracellular regulatory factors which have suppressive activity on cell growth.

Animals↗

PubMed1981

Effects of midbrain, bulbar and combined morphine microinjections and systemic injections on orofacial nociception and rostral trigeminal stimulation: independent midbrain and bulbar opiate analgesia systems?

Microinjections of 0.35 microgram and 0.7 microgram morphine were made into rat nucleus reticularis paragigantocellularis and ventral central gray respectively. When injected simultaneously, the analgesic effect on orofacial thermal pain was significantly greater than the effect of either injection alone, suggesting a summation mechanism for the two sites. No microinjection affected the threshold for aversive response to main sensory trigeminal nuclear stimulation. This threshold was, however, profoundly elevated by 10 mg/kg systemic injection.

Animals↗

PubMed1982

Reinforcing effects of brain microinjections of morphine revealed by conditioned place preference.

The previously documented place conditioning paradigm was used to study the reinforcing effects of cerebral microinjections of morphine. Rats with implanted cannulae experienced place conditioning procedures, involving morphine administration into the IV (0.5 or 10 microgram), III (10 microgram) or lateral (0.5-10 microgram) ventricles. Positive reinforcement, indicated by a significant preference for the place paired with morphine compared to the place similarly paired with control treatment, was seen in rats given 10 microgram morphine into the lateral ventricle. The rats given 10 microgram into the III ventricle also showed a preference, but the effect was not statistically significant. Positive reinforcement was subsequently demonstrated with morphine microinjections (10 microgram) into the lateral hypothalamus, periaqueductal gray or nucleus accumbens. No clear preferences were produced by morphine injections into the caudate-putamen, amygdala or nucleus ambiguus. Following the final place conditioning test, rats were re-administered the treatment dose and analgesia and body temperature were measured. All three sites associated with reinforcement evidenced hyperthermia, but only the periaqueductal gray evidenced a short-latency analgesia. Sites with null place conditioning were not associated with any major behavioral effects. Using (+) and (-)-morphine (10 microgram), it was demonstrated that only the active (-)-stereoisomer was effective in producing place preferences after injection into the periaqueductal gray. It was concluded that morphine administered directly into parts of the rat brain can produce place conditioning similar to that seen after systematically administered morphine. Morphine-produced place preference is not related to the acute depressant aspects of morphine, but may be related to the stimulant aspects.

Animals↗

PubMed1983

EEG effects of microinjection of naloxone in the region of the nucleus tractus solitarius of the rat.

In recent years, we have carried out several studies on brainstem mechanisms associated with pain modulation. Of particular interest has been the region of the nucleus tractus solitarius (NTS) at the level of the area postrema (AP) in the dorsomedial medulla. This region has been found to be rich in opiate receptors and to be involved in pain modulation. Having previously demonstrated the EEG and behavioral effects of systemic administration of morphine, we initiated the present study to determine whether microinjection of naloxone directly into the region of the NTS will modify any or all of the EEG effects induced by subsequent systemic administration of morphine. Our results have shown that microinjections of naloxone into the region of the NTS block the cortical EEG synchronizing effects of systemically administered morphine. These results indicate that morphine-induced cortical EEG synchronization is mediated through the region of the NTS. This region may, therefore, comprise part of an opiate sensitive system which can influence cortical EEG activity.

Animals↗

PubMed1984

Microinjection of vasopressin and two related peptides into the amygdala: enhancing effect on local dopamine neurotransmission.

The effects of local microinjection of Arg8-vasopressin, cyclo[Lys-Gly] and L-Pro-L-Leu-GlyNH2 (PLG) were studied on the alpha-MPT-induced disappearance of dopamine and noradrenaline in the amygdala and in a number of other brain regions. A dose-dependent increase in dopamine utilization was found in the amygdala after local microinjection of Arg8-vasopressin, cyclo[Lys-Gly] and PLG at doses of 0.1, 2.7 and 18 pmol respectively. No effects were found on noradrenaline utilization in this brain region. Using a microdissection method, it was found that cyclo[Lys-Gly] enhanced dopamine utilization in the nucl. amygdaloideus centralis, whereas the effect of PLG was mainly located in the nucl. amygdaloideus corticalis. These effects of Arg8-vasopressin, cyclo[Lys-Gly] and PLG on dopamine utilization in the amygdala are correlated with those on avoidance behavior and can be interpreted as in support of the role of dopamine as neurotransmitter involved in retrieval processes.

Amygdala↗

PubMed1985

Contralateral circling behavior induced by intranigral microinjections of taurine and GABA in rats.

Circling behavior induced by unilateral microinjections of taurine and gamma-aminobutyric acid (GABA) into the substantia nigra zona reticulata (SNR) was studied in rats. Both taurine (10-100 micrograms/rat) and GABA (30-300 micrograms/rat) induced the contralateral circling behavior in a dose-dependent manner when they were microinjected into the SNR. Neither compound induced ipsilateral circling. An ipsilateral injection of 6-hydroxydopamine (6-OHDA) into the nucleus caudatus putamen (CPU) caused a reduction of the dopamine (DA) content in the injected side and decreased the circling behavior induced by taurine but not GABA. Electrolesion of the ipsilateral nucleus ventromedialis thalami (VM) decreased both taurine- and GABA-induced circling. In contrast, only GABA-induced circling was reduced by the electrolesion of the ipsilateral nucleus parafascicularis thalami (PF). Electrolesion of the ipsilateral nucleus accumbens (ACB) did not modify the circling behavior induced by GABA, but did decrease the circling induced by taurine. Electrolesion of the ipsilateral globus pallidus (GP) or nucleus entopeduncularis (EP) did not attenuate the circling behavior induced by either taurine or GABA. These results suggest that taurine-induced circling behavior may be mediated by the nigrostriatal-nigrothalamic system, and the ACB. In contrast, the nigrothalamic system may play an important role in GABA-induced circling behavior.

Animals↗

PubMed1985

Hypertensive response to saline microinjection in the area of the nucleus tractus solitarii of the rat.

We investigated the blood pressure response elicited by microinjection of various hypertonic solutions into the area of the nucleus tractus solitarii (NTS) of the brainstem, an area rich in catecholaminergic neurons. Equiosmolar solutions of NaCl, dextrose, LiCl and KCl were employed. NaCl produced a prolonged blood pressure rise; LiCl and normal saline produced a similar rise of short duration; and KCl produced epileptic-type seizures with postictal hypertension. Dextrose had no effect and neither had NaCl microinjection in areas relatively distant from the NTS. The rise in blood pressure was not reversed by a vasopressin antagonist injected systemically, but was totally abolished by systemic alpha-adrenergic blockade with phentolamine. These findings suggest that sodium can cause hypertension by direct stimulation of the central sympathetic nervous system without participation of peripheral mechanisms such as fluid volume expansion or alteration of the vascular wall.

Animals↗