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At least 793 records · Page 44Linked to original sources

PubMed1982

Expression of a chicken lysozyme recombinant gene is regulated by progesterone and dexamethasone after microinjection into oviduct cells.

We transferred a chicken lysozyme gene recombinant by microinjection into primary cultures of chicken oviduct cells. The recombinant gene is a fusion between the lysozyme promoter, including 1.4 kb of upstream sequences, and the coding region of the gene for SV40 T antigen (plys-T). The expression of plys-T is stimulated by the steroid hormones progesterone and dexamethasone, but not by estradiol. The number of oviduct cells expressing coinjected or separately injected control genes is not increased by steroids. A deletion mutant lacking the lysozyme sequences between -161 and +15 does not express T antigen, indicating that transcription of plys-T starts within the lysozyme promoter region. By screening different cell types we found that microinjected plys-T is expressed in chicken oviduct cells but not in chicken macrophages or fibroblasts or in rat II fibroblasts.

Animals↗

PubMed1984

Microinjection of mRNA coding for an anti-Golgi antibody inhibits intracellular transport of a viral membrane protein.

Messenger RNA was prepared from a hybridoma cell line secreting a monoclonal antibody (53FC3) directed against a luminal epitope of a Golgi membrane protein (Mr = 135 kd) found in rodent cells. When this mRNA was microinjected into the cytoplasm of BHK cells, mouse IgG was seen to accumulate in the Golgi complex after 5-6 hr of incubation. No accumulation was seen in 3T3 cells which lack the epitope recognized by 53FC3. When microinjected BHK cells were infected with vesicular stomatitis virus, surface expression of the viral G protein was considerably reduced when compared with neighboring noninjected cells.

Animals↗

PubMed1984

Direct visualization of protein transport and processing in the living cell by microinjection of specific antibodies.

We have prepared polyclonal antibodies to the cytoplasmic portion of the envelope glycoprotein G of vesicular stomatitis virus (VSV) by using synthetic peptides corresponding to either the 22 or 11 ultimate carboxy-terminal residues of the G as immunogens. When antibodies to the 22 residue peptide are microinjected into monolayer baby hamster kidney cells before or shortly after infection with wild-type VSV, G protein accumulates in large intracellular patches and little G is observed in the Golgi complex or at the cell surface. In contrast, when antibodies to the 11 residue peptide are injected, no such patches are observed and G protein is seen colocalized with the injected antibody at the endoplasmic reticulum, in the Golgi complex, in transport vesicles, and at the plasma membrane. Microinjection of these antibodies does not disturb the pathway or kinetics of G-protein transport. In cells infected with a temperature-sensitive mutant of VSV, 045, the glycoprotein accumulates in the endoplasmic reticulum at 39.8 degrees C, but rapidly moves through the Golgi apparatus and then to the cell surface after a temperature shift-down to 32 degrees C. Using rhodamine-coupled antibodies to the 11 residue peptide, a microscope stage equipped for precise temperature control, and a silicon intensifier target video camera, we can visualize by video light microscopy the synchronized exocytotic transport of the G protein directly in the living cell.

Animals↗

PubMed1984

Microinjection of the oncogene form of the human H-ras (T-24) protein results in rapid proliferation of quiescent cells.

Using an E. coli expression-vector system we have efficiently produced, purified, and characterized the full-length, nonfused, protooncogenic and oncogenic (T-24) forms of the human H-ras gene product. These purified ras proteins have been introduced by microinjection into a variety of somatic cells in an effort to examine their function. Within several hours after injection of the oncogenic form of the human H-ras protein into quiescent cells, we observe dramatic morphological changes followed by transient proliferation of the cells. In contrast, microinjection of the normal, protooncogenic form of the ras protein at the same level appears to have only little effect on the cells. Additional experiments indicate that the effect of the ras protein requires entry into the cells, is temporary, is inhibited by cycloheximide or actinomycin D, and is seen only in established cell lines. This experimental approach demonstrates that the bacterially derived and purified human H-ras proteins retain their ability to function when put back into mammalian cells and furthermore, provides a novel assay for transformation induced in established cells by the human H-ras oncogene protein.

Animals↗

PubMed1995

Opiate microinjections in the locus coeruleus area of the cat enhance slow wave sleep.

The effects on sleep/wakefulness states of morphine, morphiceptin (specific mu agonist), DPDPE (delta agonist) and U-50,488H (kappa agonist) microinjections in the Locus coeruleus area (LC) were studied in cats. Morphine (0.8-1.75 nmols in 50 nl of saline) and morphiceptin (1.75 nmols) in LC significantly increased the total time spent in slow wave sleep (SWS) and the mean duration of SWS episodes. Prior naloxone administration blocked the morphine hypnogenic effects. The total time spent in SWS was unaffected by delivery of equimolar doses of DPDPE or U-50,488H in LC; however, the mean duration of the SWS episodes increased significantly after U-50,488H microinjections in LC. Thus, when acting in the LC, opiates have a SWS-enhancing effect and this effect appears to be mediated by mu receptors, although kappa receptors may have a subsidiary action.

Animals↗

PubMed1988

Performance of an automated system for capillary microinjection into living cells.

An automated capillary microinjection system for nuclear and cytoplasmic injections into cells is described. The system has been tested with samples of DNA, RNA and proteins. Movements of the capillary with precise cell positioning and time of injections are controlled by a computer. This first automated microinjection system allows injection of more than 1500 cells per hour with a minimum of practical training, volumes injected are more reproducible and cells are less damaged when compared with the standard manual injection technique. Retrieval of the injected cells is accurate to within 1% without complicated and laborious produced orientation marks on the cell support. The number of successfully injected cells is easily determined with great accuracy and the error of the statistical evaluation of the results is reduced to a minimum. Standardized procedures for pulling, handling and storage of the injection capillaries were developed.

Animals↗

PubMed1993

A remote insertion technique for intracerebral microinjections in freely moving animals.

This report describes two improvements to the typical double-cannula microinjection technique. (1) Intracerebral microinjections usually require holding the animal during the insertion of an injector through an implanted guide cannula; however, this is not necessary with the technique described. The injector is made of a long piece of fused silica capillary tubing (145 mm outer diameter x 21.2 cm) which is so small and flexible that it slips through a PE-20 tube (20 cm) that guides it into the implanted guide cannula and down to the desired brain site where it stops. (2) Connection to a microliter syringe is usually done with PE tubing which is leaky, expandable and represents a relatively large dead space that makes it difficult to deliver small, accurate volumes. This problem is avoided by making connection to the syringe via another piece of silica glass capillary tubing. Thus both the injector and its connection to the syringe are made of glass. With these modifications the injector can be inserted without touching the animal, and accurate volumes in the low nanoliter range can be delivered.

Animals↗

PubMed1995

Characterisation of an experimental model of stroke produced by intracerebral microinjection of endothelin-1 adjacent to the rat middle cerebral artery.

A novel experimental model of stroke has been developed using the powerful vasoconstrictor peptide, endothelin-1, to occlude the middle cerebral artery (MCA) of anaesthetised rats. Intracerebral microinjections of endothelin-1 were administered under stereotaxic guidance adjacent to the MCA, and after 3 days rats were perfusion fixed for histopathological determination of ischaemic brain damage. The pattern of brain damage noted using this model was similar to that reported following permanent surgical occlusion of the MCA. Brain damage was apparent in the dorsal and lateral neocortex (98 +/- 12 mm3) and striatum (32 +/- 3 mm3) ipsilateral to the insult. Rats anaesthetised with halothane and barbiturate exhibited a similar volume of brain damage. However, infarct volume increased when the duration of halothane anaesthesia was extended from 5 to 180 min post-occlusion. Neuroprotection studies demonstrated that dizocilpine (5 mg/kg, i.p.), administered 30 min prior to MCA occlusion, reduced the volume of cortical brain damage by 51% (P < 0.05) but did not alter the volume of striatal brain damage. The present results demonstrate that microinjections of endothelin-1 adjacent to the rat MCA result in a reproducible pattern of focal cerebral infarction which is sensitive to the duration of anaesthesia and can be reduced by dizocilpine.

Anesthesia↗

PubMed1992

Differential expression and stability of foreign genes introduced into human fibroblasts by nuclear versus cytoplasmic microinjection.

We have compared cytoplasmic- and nuclear-delivered, glass needle-mediated microinjection protocols for their ability to support both transient and stable phenotypic expression of reporter gene constructs in non-immortalized human skin fibroblasts cultures. Microinjection of form I (covalently closed circular, supercoiled) plasmid pMC38 DNA into the nucleus of human cells resulted in high levels of transiently expressed p110gag-myc oncoprotein as detected by immunofluorescence microscopy. Likewise, the nuclear delivery of a plasmid construct bearing the entire simian virus 40 genome induced the formation of morphologically transformed foci in approximately 6% of the recipient cell population. In contrast, the introduction of plasmid DNA by the cytoplasmic route proved virtually incapable of supporting either transient gene expression or morphological transformation. In situ autoradiography of cells injected with 3H-labelled plasmid DNA revealed that whereas the material delivered directly into the nucleus was retained by this subcellular compartment for prolonged times (greater than or equal to 48 h), the radiolabelled DNA molecules introduced via the cytoplasmic route did not reach the nucleus and appeared to be substantially degraded within 8 h following injection. These results indicate unequivocally that nuclear injection is the route of choice when monitoring foreign gene expression in human cells.

Cell Nucleus↗

PubMed1982

Delta versus mu receptors: cardiovascular and respiratory effects of opiate agonists microinjected into nucleus tractus solitarius of cats.

The cardiovascular and respiratory responses to relatively specific mu or delta agonists microinjected (0.5 microliter/kg) into the region of the nucleus of tractus solitarius (NTS) were examined in anesthetized cats. Blood pressure, heart rate, and respiratory rate were monitored for 30 min after the microinjection of opioid compounds or saline vehicle. The delta agonist, (D-Ala2,D-Leu5)-enkephalin (10-100 nmol/kg) elicited dose-dependent decreases in blood pressure, heart rate, and respiratory rate which were naloxone reversible. In contrast the mu agonists, morphine (10-54 nmol/kg) and morphiceptin (100-320 nmol/kg) had no effect on blood pressure or respiratory rate; yet, naloxone elicited pressor responses in animals pretreated with these mu agonists. A receptor-binding assay demonstrated a predominance of mu sites in the NTS. These data show that the delta opiate agonist is more effective than mu agonists in modifying cardiovascular variables in the NTS; we suggest caution in relating specific cardiovascular function to receptor subtypes defined by binding assays.

Animals↗

PubMed1990

Cardiovascular responses induced by endothelin microinjection into area postrema.

The recently described endothelium derived constricting factor endothelin (ET) is a 21 amino acid peptide which is the most potent endogenous vasoconstrictor yet described. Binding sites for this peptide have been demonstrated within the circumventricular structures of the brain. One of these structures, the area postrema (AP), has been implicated in central cardiovascular control mechanisms. We have therefore examined the effects of AP microinjection of ET on blood pressure in urethane-anaesthetised rats. Such treatment resulted in dose-dependent biphasic changes in arterial blood pressure (increases followed by decreases). Low doses of ET (0.2-1.0 pmol) induced significant increases (P less than 0.01), and high doses (5.0 pmol) significant decreases (P less than 0.01), while at intermediate concentrations (2.0 pmol) ET caused significant increases (P less than 0.05) followed by significant decreases (P less than 0.01) in mean blood pressure. Other vasoconstrictors were found to be without effect following AP administration, suggesting these changes to be the result of specific action of ET. In contrast, both ET and methoxamine had similar cardiovascular actions when microinjected into regions anatomically adjacent to the AP such as the NTS, indicating that vasoconstriction in these areas induces changes in femoral arterial blood pressure. These data suggest a specific role for ET as a chemical messenger involved in central nervous system control of the cardiovascular system within AP.

Animals↗

PubMedDate not supplied

Stimulation of food intake following opioid microinjection into the nucleus accumbens septi in rats.

The involvement of opioid peptides in the regulation of food intake has been postulated. However, it is not known how they are involved in this regulation and which brain region is responsible for the mediation of their effects. We studied the effect of a microinjection of opioid agonists and antagonists into the nucleus accumbens septi (NAS) on the food intake in rats, as this area is known to be important for motivation. Male Wistar rats were implanted stereotaxically with guide cannulae. Rats were not allowed food prior to drug treatment and solutions (1 microliter) were microinjected bilaterally. Food intake was measured throughout a 2 hr period after the drug injection. Infusions into the NAS of 2, 5 and 10 nmol of morphine, D-ala2, D-Leu5-enkephalin (DADLE), and beta-endorphin (beta E), or of 5 and 10 nmol of alpha-neoendorphin (ANEO) induced a dose-dependent increase in the food intake. Dynorphin (DYN) also increased the food intake, but only at a 10 nmol dose. The new, highly selective delta agonist D-Pen2,5-enkephalin (DPDPE) induced a dose-dependent increase in the food intake. Naloxone in doses of 2 and 10 nmol antagonized the increased food intake induced by morphine, beta E, ANEO and DYN in a dose-dependent manner, but only partly antagonized the effect of DADLE on the food intake. The selective mu-receptor antagonist beta-funaltrexamine (beta-FNA), in a dose of 5 nmol completely blocked the increase in the food intake induced by morphine but not by DADLE.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Effects of testosterone on the behavioral response to arginine vasopressin microinjected into the central gray and septum.

Arginine vasopressin (AVP) plays an important role in the control of a gonadal hormone-dependent communicative behavior in the Syrian hamster (Mesocricetus auratus) called flank marking. Previous studies have shown that gonadal hormones alter the amount of flank marking stimulated by the microinjection of AVP into the medial preoptic area-anterior hypothalamus (MPOA-AH). The purpose of the present study was to determine if testicular hormones alter the amount of flank marking stimulated by the microinjection of AVP into two other sites involved in the control of flank marking, the lateral septum-bed nucleus of the stria terminalis (LS-BNST) and the central gray. The data of the present study indicate that testicular hormones may influence the amount of AVP-stimulated marking in the central gray and LS-BNST; however, these effects are subtle and appear to occur primarily at high concentrations of AVP. When taken together with previous studies, these data indicate that gonadal hormones have greater effects on AVP-stimulated marking in the MPOA-AH than in the LS-BNST or central gray.

Analysis of Variance↗

PubMed1989

Integration and germ line transmission of foreign genes microinjected into fertilized trout eggs.

Persistence, integration into host genome, germ line transmission and expression of foreign genes microinjected into cytoplasm of fertilized rainbow trout eggs has been examined. Foreign DNA persisted as large random concatenates in approximately 50% of 6 to 12 month-old trout and exhibited a mosaic pattern between tissues. In some cases, free concatenates were observed indicating that extrachromosomal replication occurred in trout. Approximately 50% of the males had the foreign sequences in sperm DNA and all the examined animals transmitted these sequences to their progeny. The percentage of transgenic offsprings ranged from 10 to 30% and putative junction fragments were identified in Southern blot analysis in some of them. These results strongly support the hypothesis that the injected genes became integrated into the genome host, most likely after the first round of chromosomal replication. We also examined the expression of the microinjected plasmids which contained viral or mammalian promoters linked to human or rat growth hormone gene. In no case could exogenous growth hormone be detected.

Animals↗

PubMed1988

Renshaw cells are inactive during motor inhibition elicited by the pontine microinjection of carbachol.

The present study was undertaken to determine whether the postsynaptic inhibition of motoneurons that occurs following the pontine microinjection of carbachol in the decerebrate cat is due to the activity of Renshaw cells. Thirty-two out of 37 Renshaw cells (86%) were spontaneously active prior to the administration of carbachol, whereas only 2 out of 13 Renshaw cells (15%) discharged during carbachol-induced motor inhibition. In addition, discrete inhibitory synaptic potentials were observed in 33% of the Renshaw cells from which intracellular recordings were obtained after carbachol administration, indicating that these cells were actively inhibited. The finding that a population of Renshaw cells, which inhibit motoneurons, were themselves inhibited during a period of profound motoneuron inhibition was quite unexpected. These results support the conclusion that Renshaw cells are not the inhibitory interneurons that are responsible for the powerful inhibition of motoneurons that occurs following the pontine microinjection of carbachol.

Action Potentials↗

PubMed1989

Cardiovascular effects of increasing sodium chloride concentrations microinjected into the nucleus tractus solitarii of the rat.

Unilateral microinjection of increasing sodium chloride concentrations (154.0-256.6 mM) into the nucleus tractus solitarii (NTS) of urethane-anesthetized rats evoked hypotension and bradycardia. The effect was elicited only in a restricted area of the mediocaudal NTS. Microinjection of demineralized water, an isotonic sucrose solution and artificial cerebrospinal fluid were without effect. The possible existence of sodium-sensitive neurons in the NTS is discussed.

Animals↗

PubMed1990

Catalepsy induced by carbachol microinjected into the pontine reticular formation of rats.

Rats were implanted stereotaxically with permanent guide tubes aimed at the pontine reticular formation. Carbachol was microinjected in the awake, freely moving rats. Catalepsy was evaluated with the horizontal bar test. Intense and long-lasting, dose-dependent catalepsy was observed following microinjections of 2-8 micrograms of carbachol. Pretreatment with intraperitoneally administered scopolamine significantly reduced the cataleptogenic effect of carbachol. These results show that the pontine reticular formation is part of the neuroanatomical substrate of catalepsy induced by cholinergic drugs.

Animals↗

PubMed1991

Carbachol microinjections in the mediodorsal pontine tegmentum are unable to induce paradoxical sleep after caudal pontine and prebulbar transections in the cat.

In 7 cats, total transections of the brainstem at the caudal pontine or the prebulbar level led to preparations which presented neither behavioral nor electrophysiological signs of paradoxical sleep (PS) throughout their survival periods (17-30 days). Carbachol microinjections in the mediodorsal pontine tegmentum (MDPT), which induced PS in the intact cat, were no longer able to induce it in the transected animals. Rapid eye movement (REM) and pontogeniculo-occipital (PGO)-like bursts were evoked by carbachol microinjections in the pontine magnocellular tegmental field (FTM) of cats transected at the prebulbar level, as in the intact cat. Only REM bursts were obtained by the same injections in caudal pontine transected cats. It is concluded that (1) the pons is insufficient to generate PS; (2) complex reciprocal interactions with the medulla are necessary for the generation of this state of sleep; and (3) the production of long REM and PGO bursts is controlled by the caudal pontine tegmentum.

Animals↗