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PubMed1990

Cardiovascular effects of L-glutamate and tetrodotoxin microinjected into the rostral and caudal ventrolateral medulla in normotensive and spontaneously hypertensive rats.

The purpose of this study was to compare the responsiveness of the rostral and caudal ventrolateral medulla in spontaneously hypertensive (SH) and normotensive Wistar-Kyoto (WKY) rats to microinjection of L-glutamate, and to estimate tonic output of these areas by microinjecting the neurotoxin tetrodotoxin. Rats were anesthetized with 1.25 g/kg urethane s.c., implanted with arterial (femoral) and venous (femoral) catheters, artificially ventilated and paralyzed with gallamine triethiodide (10 mg/kg). Using a ventral approach to the brainstem, the mean arterial pressure and heart rate responses to microinjection (30 nl) of L-glutamate (1, 10 and 100 mM) and tetrodotoxin (10 microM) into the rostral and caudal ventrolateral medulla were compared in SH (n = 7) and WKY (n = 7) groups. Microinjection of L-glutamate into the rostral ventrolateral medulla produced equivalent increases in mean arterial pressure (maximum +33 +/- 3 and +36 +/- 6 mm Hg, SH and WKY groups respectively) and minimal changes in heart rate. Similar administration of L-glutamate into the caudal ventrolateral medulla caused decreases in mean arterial pressure and heart rate; changes in mean arterial pressure were significantly greater in the SH group than in the WKY group (-52.3 +/- 2.9 mm Hg for SH, -22.6 +/- 2.6 mm Hg for WKY). Bilateral microinjection of tetrodotoxin into the caudal ventrolateral medulla produced significantly larger increases of mean arterial pressure in WKY rats (+8 +/- 4 vs +46 +/- 8 mm Hg for SH vs WKY). These data indicate that SH rats may have a lower tonic activity of neurons in the caudal ventrolateral medulla, resulting in a lower restraining influence on sympathetic outflow in the SH rat.

Action Potentials↗

PubMed1989

Talin dynamics in living microinjected nonmuscle cells.

To investigate the role of talin in the anchoring of actin-containing stress fibers to the cell membrane of nonmuscle cells, a fluorescent analog of the adhesion plaque protein talin was developed, characterized, and microinjected into living cells. Purified chicken gizzard talin was covalently labeled with the fluorescent dye lissamine rhodamine B sulfonyl chloride. The fluorescently labeled protein was then chromatographed on Sephadex G-25 and DEAE-cellulose in order to remove free dye and denatured protein. The fluorescent talin was able to bind purified vinculin and was localized in adhesion plaques, membrane ruffles, microspikes, and polygonal networks in acetone-permeabilized nonmuscle cells. In cells that were double-stained with fluorescent talin and an affinity-purified anti-talin antibody, a one-to-one correspondence of adhesion plaque staining was seen. Living epithelial cells (PtK2) were microinjected during interphase with fluorescent talin. Computer-enhanced video microscopy was used to document adhesion plaque dynamics such as 1) changes in plaque shape, 2) alterations in plaque positions, and 3) the appearance, growth, and dissolution of plaques. In cells that were followed during mitosis, the adhesion plaques disappeared during cell rounding and then subsequently reappeared upon spreading of the two daughter cells. Treatment of microinjected cells with DMSO in order to disassemble stress fibers resulted in an altered localization of the fluorescent talin. Upon recovery of the cell from the drug, the talin was visualized in its characteristic submembraneous position. These results are the first to document the role and distribution of talin in dynamic processes occurring in living microinjected nonmuscle cells.

Animals↗

PubMed2006

Microinjection of carbachol in the supramammillary region suppresses CA1 pyramidal cell synaptic excitability.

Previous studies have established that the posterior hypothalamus-supramammillary (SUM) region is involved in the control of the hippocampal theta rhythm and also modulates the synaptic excitation of hippocampal neurons. Particularly, the medial but not lateral SUM region mediates reticular stimulation-induced suppression of CA1 pyramidal cell synaptic excitation to Schaffer collateral stimulation. In the present study using urethane anesthetized rats, we have investigated the effect of direct chemical stimulation of the posterior hypothalamus-SUM region on CA1 pyramidal cell excitability. It was observed that microinjection of the cholinergic muscarinic receptor agonist, carbachol (0.1 microl, concentration of either 0.0052, 0.156, or 0.625 microg/microl), evoked concentration-dependent suppression of CA1 pyramidal cell excitability that was dissociated from theta activation. Further, carbachol microinjection preferentially recruited the lateral SUM region when compared with the medial SUM and the posterior hypothalamic regions. In this context, the shortest latencies to suppression at the lowest concentration of carbachol and the strongest suppression at higher concentrations were observed with lateral microinjections. The carbachol-induced suppression was attenuated by inactivation of the medial septal region by microinjection of procaine (0.5 microl, 20% w/v). These results underscore a possible role for cholinergic mechanisms in the lateral SUM region in modulation of CA1 pyramidal cell synaptic excitation via the medial septal region. Furthermore, the present findings when juxtaposed with the medial SUM mediation of reticularly-elicited suppression suggest a medial-lateral topographic organization of the SUM region in modulation of CA1 excitability.

Acetylcholine↗

PubMed1991

Fertilizing capacity of epididymal and testicular spermatozoa microinjected under the zona pellucida of the mouse oocyte.

Mouse spermatozoa were obtained from the testis and caput, corpus, and cauda epididymis incubated for 2 h in capacitation medium and a single spermatozoon from the capacitated samples microinjected into the pervitelline space of mature mouse oocytes. Spermatozoa from the testis were unable to fertilize oocytes and few spermatozoa from the caput were capable of fertilization (0-7% depending on the method of preparation). Similar fertilization rates (30-45%) were obtained with spermatozoa with forward progressive motility from the proximal and distal corpus and the cauda epididymis, but those that were vibratory or with local circular motility had significantly reduced fertilizing capacity (6-10%). The capacity of spermatozoa from the different regions of the testis and epididymis to bind to zona-free oocytes followed the same pattern as fertilization rate after microinjection. There was a progressive increase in acrosome reactions after 2 h incubation in capacitation medium in samples obtained from the testis to the cauda epididymis. Maturation of the capacity to bind to the perivitelline membrane and to develop forward progressive motility, rather than the capacity to acrosome react, appears to govern the fertilization of oocytes in which the zona has been bypassed by microinjection. These characteristics are obtained in the proximal segment of the corpus. However, there was evidence that the embryo developmental capacity of oocytes fertilized by spermatozoa from the higher segments of the epididymis is reduced, particularly in oocytes fertilized by caput spermatozoa. These observations suggest that sperm microinjection may have only a limited benefit for improving fertilization rates for men with high epididymal obstructive azoospermia.

Acrosome↗

PubMed1993

System for quantitation of gene expression in single cells by computerized microimaging: application to c-fos expression after microinjection of anti-casein kinase II antibody.

A system which allows sensitive and fast automated analysis of weakly labeled fluorescent specimens is described. It is tested in the analysis of c-fos expression stimulated by fetal calf serum and calibrated by quantitation of defined solutions injected into cells with the automated microinjection system. Low light level imaging technology combined with quantitative image processing methods and computer control of the hardware allows fully automated analysis of fluorescent molecules in single living or fixed cells. Reliable methods for subtraction of fluorescent background and automated identification of objects of interest in double-stained cells are described. The accuracy of quantitation is considerably improved by normalizing the fluorescence intensities of respective fluorophores in the same object by the method of ratio imaging. The error rate in determining the relative protein content in single cells is less than 15%. The method is applied to microinjection studies with a monoclonal antibody against casein kinase II subunit beta. Microinjection of this antibody into synchronized cells specifically inhibits c-fos expression stimulated by fetal calf serum. In combination with the computer-automated capillary microinjection system, the technique will become a useful tool in experiments requiring quantitative single cell analysis.

Antibodies↗

PubMed1993

Microinjected catalytic subunit of cAMP-dependent protein kinase induces apoptosis in myeloid leukemia (IPC-81) cells.

Rat IPC-81 promyelocytic leukemia cells responded to cAMP analog by undergoing apoptotic cell death both when anchored to fibronectin and when free in the medium. The protein kinase C stimulator 12-O-tetradecanoylphorbol 13-acetate enhanced the anchoring to substratum without impeding cAMP-induced cell death. The immobilized cells could be microinjected. This made it possible to study the effect on apoptosis of microinjected catalytic (C alpha) and regulatory (RI alpha D199) subunits of cAMP-dependent protein kinase as well as of phosphatase inhibitors. Microinjection of C alpha reproduced the morphological effects of cAMP, including nuclear fragmentation. RI alpha D199 blocked the effect of C alpha. Injection of microcystin-LR, which inhibits protein phosphatases 1 and 2A, led to pronounced apoptoid changes of the leukemia cells, but failed to produce nuclear fragmentation. Microinjection of peptide inhibitors ("inhibitor 1" and "inhibitor 2") specific for phosphatase 1 had no effect on cell morphology. The failure of the phosphatase inhibitors to reproduce completely the effect of the C subunit underscores the specificity of action of the latter.

Animals↗

PubMed2002

Microinjected anti-actin antibodies decrease gap junctional intercellular commmunication in cultured astrocytes.

The purpose of the present study was to investigate the influence of the actin cytoskeleton on gap junctional intercellular communication (GJIC) in cultured astrocytes. This report describes an decrease of GJIC following microinjection of anti-actin antibodies or cytochalasin D treatment. Dye transfer of microinjected neurobiotin was used to assay gap junctional permeability in cultured astrocytes. Besides this translocation of connexins to the plasma membrane we investigated subsequent anti-actin antibody injection. While control cultures exhibited intensive dye spreading of microinjected neurobiotin, GJIC was impaired by microinjection of anti-actin antibodies. Additionally, impaired GJIC was observed after cytochalasin D treatment for 15 min. After the drug had been washed out, a recovery of GJIC was achieved. Cultured astrocytes exhibited a prominent actin cytoskeleton, with strong staining of actin filaments at the plasma membrane. Confocal laser scanning microscopy revealed an impaired translocation of Cx 43 from the Golgi apparatus to the cell membrane of cell processes following anti-actin antibody injection. These results suggest that the morphological integrity of microfilaments seems to be fundamental for GJIC, probably by means of associations among actin filaments, actin binding proteins, and Cx 43 at the plasma membrane or indirectly through the transport of connexins from the cytoplasm to the cell membrane.

Actin Cytoskeleton↗

PubMed1996

Effects of morphine microinjections into the trigeminal sensory complex on the formalin test in the rat.

The aim of this study was to find out whether morphine locally applied into the different subnuclei of the spinal trigeminal nucleus could impair the behavioral response evoked by a tonic nociceptive stimulus. Microinjections of morphine were performed unilaterally in rats through a chronically implanted tube. The duration of the formalin-induced biphasic rubbing activity was used as a measure of nociception. Morphine, 0, 2, 7, or 14 microg, in 0.2 microl of saline were microinjected 10 min before a subcutaneous injection of formalin (1.5%) in the ipsilateral upper lip. Morphine microinjections into subnucleus oralis (Sp5O) and subnucleus caudalis (Sp5C) induced a significant decrease in rubbing duration of either the early or late phase of the response or both. The early response to formalin was depressed only after morphine injection into Sp5O. The late response was depressed by microinjections into both Sp5O and Sp5C. No significant effect was observed in subnucleus interpolaris (Sp5I). These results are further evidence for the role played by Sp5C in orofacial nociception. They also give support to the involvement of the Sp5O in perioral nociceptive mechanisms of short duration.

Analgesics, Opioid↗

PubMed1996

Toxicity of 4-chloroaniline in early life-stages of zebrafish (Brachydanio rerio): I. cytopathology of liver and kidney after microinjection.

In addition to survival and hatching parameters, cytological alterations in liver and kidney of 4- and 6-d old zebrafish larvae (Brachydanio rerio) following single microinjection of fertilized eggs at the germ-ring stage with 5, 12.5, and 25 ng 4-chloroaniline/egg were investigated by means of electron microscopy. Whereas survival remained unaffected, microinjection with 4-chloroaniline disturbed hatching of larvae. Hatching was delayed by microinjection of 12.5 ng 4-chloroaniline/egg and above when compared to controls. Cytological investigations revealed ultrastructural changes in both liver and kidney in a dose- and time-dependent fashion. In the liver, major cytopathological changes included fenestration, fragmentation, and vesiculation of the rough endoplasmic reticulum, proliferation of atypical mitochondria, and atypical lysosomes. Furthermore, myelin whorls, lipid inclusions, and cholesterol crystals were increased, whereas glycogen stores were reduced. Renal tubular cells displayed altered brush borders, proliferation of nucleoli, atypical mitochondria, fenestrated, fragmented, and vesiculated RER cisternae, as well as giant lysosomes. Most of these effects indicate cellular dysfunction (e.g., disturbance of lipid metabolism in the liver), whereas others illustrate general cellular stress-responses to chemical aggression. Comparisons of results with those of previous studies based on conventional fish exposure prove the suitability and sensitivity of microinjection bioassays with zebrafish eggs as an alternative to conventional early life-stage tests.

Aniline Compounds↗

PubMed1992

Axoplasmic transport of horseradish peroxidase in single neurons of the dorsal root ganglion studied in vitro by microinjection.

The dependence of anterograde axoplasmic transport on cytoskeletal components was investigated using microinjection of horseradish peroxidase (HRP) into the somata of chick dorsal root ganglion cells in vitro. Microinjected HRP was transported anterogradely in the neurites and their branches; this transport was disturbed by colchicine in a drug-dependent and time-dependent manner. Cytochalasin B, a drug that depolymerizes actin, did not inhibit the transport of HRP, despite the formation of local swellings in neurites. The microinjection of polyclonal antibodies directed against tubulin and monoclonal antibodies (mAbs) against 200-kDa neurofilaments disturbed the axoplasmic transport of co-injected HRP, which then exhibited an irregular and discontinuous distribution in the axonal branches. The transport of HRP became discontinuous after the injection of anti-tubulin antibodies and led to the formation of globular deposits of HRP. Polyclonal antibodies against actin and mAbs to 160-kDa and 68-kDa neurofilaments seemed to have no effect on the axoplasmic transport of co-injected HRP. Microinjection of antibodies against tubulin induced formation of perinuclear bundles consisting of cytoskeletal components. The transport of HRP thus appears to be regulated by an intact microtubular system and cross-linker components (200-kDa neurofilaments) of the cytoskeleton. Actin and most intermediate filament proteins do not seem to play an essential role in the transport of HRP.

Animals↗

PubMed1990

Fertilization of human oocytes by microinjection of human sperm aspirated from the caput epididymidis of an individual with obstructive azoospermia.

Aspiration of spermatozoa from the caput region of the epididymis was performed, due to obstructive azoospermia. When all inseminated oocytes failed to fertilize in vitro, immotile spermatozoa were microinjected into the perivitelline space to achieve fertilization. Of six oocytes microinjected, one exhibited two normal pronuclei and two polar bodies and was subsequently transferred to a fallopian tube of the wife. One oocyte arrested at syngamy, while two additional oocytes cleaved to four cells each. The remaining two were damaged during microinjection. Although the patient did not conceive, this report provides evidence that (1) spermatozoal motility is not required for successful pronuclear formation when microinjection is performed, and such immotile sperm are not necessarily "dead," and (2) this further corroborates that caput epididymal spermatozoa can participate in the subzonal events of fertilization and that, in the human, exposure of spermatozoa to the distal epididymal milieu may not be a requisite to achieve fertilizing capacity.

Adult↗

PubMed1977

Chemically facilitated microinjection of proteins into intact monolayers of tissue culture cells.

Microinjection of physiologic quantities of macromolecules into tissue culture cells can facilitate the study of the biological effects of such macromolecules. In this communication, we describe a chemical technique which can be used to microinject proteins into monolayers of intact cells. Protein is loaded into erthrocyte ghosts, and the ghosts are then fused to the monolayer with polyethylene glycol 1000. Receipient cells can be injected with an efficiency of greater than 90% and contain an average of 3.8 X 10(6) microinjected molecules per cell. This technique circumvents certain problems encountered in virus-induced microinjection.

Cell Fusion↗

PubMedDate not supplied

Glass needle-mediated microinjection of macromolecules and transgenes into primary human mesenchymal stem cells.

Human mesenchymal stem cells (hMSCs) are multipotent cells that can differentiate into various tissue types, including bone, cartilage, tendon, adipocytes, and marrow stroma, making them potentially useful for human cell and gene therapies. Our objective was to demonstrate the utility of glass needle-mediated microinjection as a method to deliver macromolecules (e.g. dextrans, DNA) to hMSCs for cell and molecular biological studies. hMSCs were isolated and cultured using a specific fetal bovine serum, prescreened for its ability to promote cell adherence, proliferation, and osteogenic differentiation. Successful delivery of Oregon Green-dextran via intranuclear microinjection was achieved, yielding a postinjection viability of 76 +/- 13%. Excellent short-term gene expression (63 +/- 11%) was achieved following microinjection of GFP-containing vectors into hMSCs. Higher efficiencies of short-term gene expression ( approximately 5-fold) were observed when injecting supercoiled DNA, pYA721, as compared with the same DNA construct in a linearized form, YA721. Approximately 0.05% of hMSCs injected with pYA721 containing both the GFP and neomycin resistance genes formed GFP-positive, drug-resistant colonies that survived >120 days. Injection of linearized YA721 resulted in 3.6% of injected hMSC forming drug-resistant colonies, none of which expressed GFP that survived 60-120 days. These studies demonstrate that glass needle-mediated microinjection can be used as a method of delivering macromolecules to hMSCs and may prove to be a useful technique for molecular and cell biological mechanistic studies and future genetic modification of hMSCs.

Cells, Cultured↗

PubMed2000

Ultrastructural analysis of nucleolar transcription in cells microinjected with 5-bromo-UTP.

In situ sites of nucleolar transcription in cells microinjected with 5-bromo-UTP (BrUTP) were visualized at an ultrastructural level. After injection the cells were maintained for 4-90 min at 37 degrees C, fixed, and embedded in LR White resin. Postembedding immunoelectron microscopic visualization with colloidal gold has been used for localizing both Br-labeled precursor incorporated into pre-rRNA and different nucleolar transcription or processing factors. This high resolution approach allowed us to identify significant signal as early as after 4-min incubation periods following BrUTP microinjection. It revealed the dense fibrillar component (DFC) as being the first nucleolar compartment labeled with anti-bromodeoxyuridine antibody. Moreover, RNA polymerase I, nucleolar transcription factor UBF, and fibrillarin were also detected almost exclusively in this same nucleolar compartment. From 30 min onward, following microinjection, Br-labeled rRNA occurred also in the granular component. The results indicate that the DFC is the site of pre-rRNA transcription and of initial steps of pre-rRNA processing. Moreover, it demonstrates that BrUTP microinjection followed by postembedding detection of Br-labeled RNA is a useful technique for high resolution studies of structure-function associations in the nucleolus.

Cell Nucleolus↗

PubMed1998

Responses of vestibular neurons to arginine vasopressin microinjection.

The aim of the present research was to evaluate the effects induced by arginine vasopressin (VP) microinjection on the electrical activity of single vestibular neurons. Experiments were performed on anaesthetized guinea-pigs in which the spontaneous and the evoked electrical activity of vestibular neurons were recorded before and after intranuclear VP microinjection (0.25.10(-5) pg VP in 0.25 microliter NaCl 0.9% solution). Results showed that VP microinjection affects the spontaneous as well as the evoked vestibular neuron activity. More precisely, 60% of 30 tested neurons were inhibited, 30% were excited and the remaining 10% were unaffected by VP microinjection. The changes in neuronal activity reported above were attributed to a direct action exerted by the polypeptide on vestibular complex neurons. The possible role played by VP in the mechanisms of postural control exerted by the vestibular system was considered as well.

Animals↗

PubMed2005

Aftereffects of microinjections of neurotensin into the substantia nigra of the brain on conditioned motor responses in rats with lesions to serotoninergic neurons.

The effects of neurotensin microinjections into the substantia nigra of the brain on the performance of motor reactions in response to positive (food reinforcement) and negative (unreinforced) conditioned signals were studied, along with the characteristic aftereffects of these microinjections in rats with lesions to serotoninergic neurons in the dorsal cervical nucleus. Lesions were produced by local administration of the selective neurotoxin 5,7-dihydroxytryptamine. Microinjections of neurotensin were found to weaken the disinhibitory effects of neurotoxin on extinction of intersignal responses and extinction of conditioned motor reactions in response to presentation of negative stimuli, but to have no marked effect on the performance of reactions evoked by positive conditioned signals. Changes persisted into subsequent experiments without peptide microinjections. Measurements of the level of preference of the animals for one quadrant of the experimental chamber showed that operated rats avoided occupying the test quadrant and preferred to locate themselves in the opposite quadrant. Neurotensin released this effect of the neurotoxin. These results lead to the conclusion that these behavioral effects of neurotensin are associated with its normalizing influences on the motivational-emotional state of the animals with lesions to serotoninergic neurons and may also be due to the formation in the animals of a contextual conditioned emotional state, which aids optimization of the adaptive functions of the brain.

5,7-Dihydroxytryptamine↗

PubMed1987

Analgesic effects of serotonin microinjection into nucleus raphe magnus and nucleus raphe dorsalis evaluated by the monosodium urate (MSU) tonic pain model in the rat.

The effects of 5-hydroxytryptamine (5-HT) microinjection into the nucleus raphe magnus (NRM) and the nucleus raphe dorsalis (NRD) on tonic pain were studied using the monosodium urate (MSU) tonic pain model in the rat. For the NRM, 5-HT microinjection produced significant analgesic effects, which were antagonized by systemic naloxone administration and also by subsequent microinjection of naloxone into the NRM. For the NRD, systemic naloxone administration did not antagonize these analgesic effects, although 5-HT microinjection produced significant analgesic effects. Therefore, as far as tonic pain is concerned, it was suggested that neural transmission mediated by 5-HT in the NRM and NRD plays an antinociceptive action, but via different neural mechanisms.

Analgesia↗

PubMed1988

GABAergic modulation of nociceptive threshold: effects of THIP and bicuculline microinjected in the ventral medulla of the rat.

Neurons of the nucleus raphe magnus (NRM) and nucleus reticularis gigantocellularis pars alpha (NGCp alpha) have been implicated in the regulation of nociceptive threshold and production of antinociception. Previous studies have shown that the activity of these neurons is modulated by noradrenergic, cholinergic and serotonergic afferents. The present study examined whether these neurons are additionally subject to regulation by a GABAergic input. Microinjection of the GABAA receptor agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP; 0.3 or 1.0 microgram) in the NRM or NGCp alpha significantly decreased tail flick latency (TFL) and increased responsiveness to noxious pinch. Hot plate latency (HPL) was not affected by microinjection of 0.3 microgram THIP. Although HPL was increased after microinjection of 1.0 microgram THIP, this effect may reflect motoric disturbances. In contrast to the hyperalgesia produced by THIP, microinjection of the GABAA receptor antagonist bicuculline methiodide (0.04 or 0.1 microgram) produced a small, but significant increase in TFL. Responsiveness to noxious pinch and HPL were not affected by either dose. These findings indicate that neurons of the NRM or NGCp alpha involved in the regulation of nociceptive threshold are subject to an inhibitory GABAergic input mediated by a GABAA receptor. However, in contrast to previously described inhibitory inputs, the GABAergic influence does not appear to be tonically active to a substantial extent in the unanesthetized rat.

Animals↗