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PubMedDate not supplied

Cardiovascular responses to medullary microinjections of opiate agonists in urethane-anesthetized rats.

Pressor (VLPA) and depressor (VLDA) areas of the ventrolateral medulla were identified by microinjections of L-glutamate in urethane-anesthetized rats. Cardiovascular effects of opiate agonists microinjected into the same sites were then studied. Agents used to stimulate mu, delta, sigma, kappa, and beta-endorphin (epsilon) receptors were morphiceptin, D-Ala2-D-Leu5-enkephalin, N-allyl-normetazocine, dynorphin, and beta-endorphin, respectively. Opiate receptor stimulation in VLPA decreased blood pressure (BP) and heart rate (HR), while in VLDA it increased BP and HR. Thus, it is the site of injection rather than the type of opiate receptor that determines cardiovascular responses. Naloxone, an opiate antagonist, reversed and prevented these responses. Abolition of cardiovascular responses by spinal transection at the C1 level indicated that the sympathetic nervous system mediated these responses. The following mechanism is proposed for these actions of opiates: Cell bodies in VLPA, but not in VLDA, project to the intermediolateral cell column of the spinal cord. Opiates inhibit VLPA and lower BP and HR by decreasing sympathetic outflow. Opiate-induced inhibition of VLDA, which has an inhibitory effect on VLPA, results in an increase in BP and HR.

Animals↗

PubMed1999

Cardiovascular responses produced by microinjection of serotonin-receptor agonists into the paraventricular nucleus in conscious rats.

Activation of serotonin (5-hydroxytryptamine, 5-HT) receptors in the brain produces cardiovascular responses by altering autonomic outflow. The paraventricular nucleus (PVN) contains a modest density of 5-HT receptors and has connections to autonomic centers. Experiments were designed to determine whether cardiovascular responses were produced by the administration of 5-HT2- and 5-HT1A-receptor agonists into the PVN of conscious rats. The microinjection of the 5-HT2-receptor agonist DOI [(+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane HCl] into the PVN produced dose-dependent (1-10 nmol) increases in heart rate and blood pressure; the peak responses were +39 +/- 10 beats/min and +6 +/- 2 mm Hg, respectively. Both responses were blocked by the concomitant administration of the selective 5-HT2-receptor antagonist LY53857 into the PVN. By contrast, the microinjection of the selective 5-HT1A-receptor agonist R(+)-8-OH-DPAT [R(+)8-hydroxy-2-(di-n-propylamino) tetralin HBr; 1-10 nmol] into the PVN did not affect blood pressure or heart rate. These data suggest that 5-HT neurons projecting from the raphe nuclei to or near the PVN can participate in the central control of the cardiovascular system by way of 5-HT2 receptors. Apparently 5-HT neurons terminating in the PVN can increase blood pressure and heart rate and produce sympathoadrenal activation, metabolic and hormonal responses consistent with those observed in several different stress paradigms.

8-Hydroxy-2-(di-n-propylamino)tetralin↗

PubMed2005

Pushing the envelope: microinjection of Minute virus of mice into Xenopus oocytes causes damage to the nuclear envelope.

Parvoviruses are small DNA viruses that replicate in the nucleus of their host cells. It has been largely assumed that parvoviruses enter the nucleus through the nuclear pore complex (NPC). However, the details of this mechanism remain undefined. To study this problem, the parvovirus Minute virus of mice (MVM) was microinjected into the cytoplasm of Xenopus oocytes and a transmission electron microscope was used to visualize the effect of the virus on the host cell. It was found that MVM caused damage to the nuclear envelope (NE) in a time- and concentration-dependent manner. Damage was predominantly to the outer nuclear membrane and was often near the NPCs. However, microinjection experiments in which the NPCs were blocked showed that NE damage induced by MVM was independent of the NPC. To address the question of whether this effect of MVM is specific to the NE, purified organelles were incubated with MVM. Visualization by electron microscopy revealed that MVM did not affect all intracellular membranes. These data represent a novel form of virus-induced damage to host cell nuclear structure and suggest that MVM is imported into the nucleus using a unique mechanism that is independent of the NPC, and involves disruption of the NE and import through the resulting breaks.

Animals↗

PubMed1990

Microinjected fluorescent phalloidin in vivo reveals the F-actin dynamics and assembly in higher plant mitotic cells.

Endosperm mitotic cells microinjected with fluorescent phalloidin enabled us to follow the in vivo dynamics of the F-actin cytoskeleton. The fluorescent probe immediately bound to plant microfilaments. First, we investigated the active rearrangement of F-actin during chromosome migration, which appeared to be slowed down in the presence of phalloidin. These findings were compared with the actin patterns observed in mitotic cells fixed at different stages. Our second aim was to determine the origin of the actin filaments that appear at the equator during anaphase-telophase transition. It is not clear whether this F-actin is newly assembled at the end of mitosis and could control plant cytokinesis or whether it corresponds to a passive redistribution of broken polymers in response to microtubule dynamics. We microinjected the same cells twice, first in metaphase with rhodamine-phalloidin and then in late anaphase with fluorescein isothiocyanate-phalloidin. This technique enabled us to visualize two F-actin populations that are not co-localized, suggesting that actin is newly assembled during cell plate development. These in vivo data shed new light on the role of actin in plant mitosis and cytokinesis.

Actin Cytoskeleton↗

PubMed2004

Stimulation of luteinizing hormone subunit gene expression by pulsatile intracerebroventricular microinjection of galanin in female rats.

Although galanin, which exerts its effects both at the hypothalamic and pituitary level, has been implicated as an important neuroendocrine regulator of hypothalamic-pituitary-gonadal axis activity, there is a lack of data concerning its involvement in the regulation of gonadotropin subunit gene expression. To elucidate whether galanin can influence luteinizing hormone (LH) subunit mRNA content, as well as affect gonadotropin-releasing hormone (GnRH) receptor activity, a model based on pulsatile (one pulse per hour over 5 h) galanin (1 nM) microinjections directly into the third cerebral ventricle of ovariectomized (OVX) and/or oestrogen/progesterone-pretreated rats was used. Furthermore, to determine galanin effects on GnRH-induced LH subunit mRNA synthesis, a cocktail of 1 nM GnRH and 1 nM galanin was coadministered in a pulsatile manner to OVX/steroid primed rats. Subsequently, to obtain data concerning the role of galanin receptors in the regulation of pituitary alpha (common to LH, follicle-stimulating hormone, thyroid-stimulating hormone) and LHbeta subunit gene expression, OVX/oestrogen/progesterone rats received microinjections of 1 nM of the receptor antagonist galantide and 1 nM of galanin. In this case, both substances were administered separately, with a 30 min lag, according to which each galantide pulse always preceded a galanin pulse. Northern-blot analysis revealed that intracerebroventricular pulsatile galanin injections were effective in stimulation of both alpha and LHbeta subunit mRNA levels and that this effect was apparently steroid-dependent. Moreover, galanin also up-regulated GnRH receptor functional parameters (affinity and maximum binding capacity) but was ineffective in potentiating GnRH-induced accumulation of both subunit mRNAs. The results from the study also indicate that galanin acts through its own receptor(s) because a receptor antagonist, galantide, significantly reduced the stimulatory effect exerted by galanin on the expression of both LH subunit genes in vivo.

Animals↗

PubMed1987

Direct microinjection of substances in penetration-sensitive embryos.

We have modified an electrode holder into an oil-pressure holder by fitting an internal platinum heating filament into the holder. This design enables easy preparation, mounting, and exchange of micropipettes. Micropipettes with very thin tips (outer diameter less than 0.1 micron) can be used, as the system allows the use of back-filled pipettes. Therefore, successful microinjections in penetration-sensitive embryos are possible. The characteristics of the system are described, and its advantages and disadvantages, as compared to other microinjection systems, are discussed.

Animals↗

PubMed1983

Subcellular distribution in Xenopus laevis oocytes of a microinjected poly(A)-binding protein from Artemia salina gastrulae.

The poly(A)-binding protein P38 of non-polysomal mRNP from Artemia salina gastrulae was labelled by reductive methylation and microinjected into the cytoplasm of Xenopus laevis oocytes. The labelled protein has a half-life of approximately 20 h and accumulated in the nucleus of the oocyte. The kinetics of accumulation reached a plateau at about 15 h after microinjection. P38 accumulates in the nucleus to a final concentration 3.15 times higher than that reached by free diffusion. This fact suggests that P38, a cytoplasmic poly(A)-binding protein, might also play some role in the nucleus of the cell.

Animals↗

PubMed1987

Microinjection of kainic acid into the rostral ventrolateral medulla causes hypertension and release of neuropeptide Y-like immunoreactivity from rabbit spinal cord.

Microinjections of kainic acid were made into the pressor area of the rostral ventrolateral medulla oblongata of anaesthetized rabbits, in the region of the C1 adrenaline-containing neurons. Over the 65 min following the microinjection, there was a significant increase in blood pressure, as well as an increase in the release of neuropeptide Y-like immunoreactivity into the spinal subarachnoid space. These data provide evidence for a functional bulbospinal neuropeptide Y-containing projection which may be responsible for mediating the pressor effects of stimulation of the rostral ventrolateral medulla.

Animals↗

PubMed1995

Evolution of dose-related cardiovascular responses to L-glutamate microinjected into the nucleus tractus solitarii after removal of the nodose ganglion in rat.

1. In this study, we sought to determine if sensitivity to dose-related changes in arterial pressure (AP) and heart rate (HR) elicited by microinjection of L-glutamate (L-GLU) into the nucleus tractus solitarii (NTS) evolves after deafferentation in adult male Sprague-Dawley rats. 2. Twelve rats served as unoperated controls; and eight were studied 5 days, eleven 10 days and nine 15 days after ganglionectomy. Each rat was anaesthetized (halothane 1.5-2.0%) and cannulated for recording AP and HR. Vehicle or L-GLU (3, 30, 75, 150, 300 and 1500 pmole/50 nL) was microinjected into the right and left NTS. Injection sites were confirmed histologically. 3. In control animals and in animals 5 days and 15 days after ganglionectomy, the lowest dose of L-GLU that produced a significant fall of AP was 30 pmole. The threshold dose with injections on the lesioned side 10 days after ganglionectomy was 3 pmole. In rats studied at 10 days, but not in control, 5 or 15 day rats, dose-related responses for injections made on the left were shifted significantly to the left of those made on the right (P < 0.05). 4. In control, 5 and 15 day rats there were no significant differences between dose-related responses elicited from right and left NTS. Responses of HR did not differ between groups of animals or sides of injection. 5. These results suggest that supersensitivity to L-GLU evolves after nodose ganglionectomy and support a role for L-GLU as a transmitter of cardiovascular afferents in the NTS.

Anesthesia↗

PubMed1987

Lack of evidence for increased descending inhibition on the dorsal horn of the rat following periaqueductal grey morphine microinjections.

1 Recordings were made from 18 neurones in the dorsal horn of the rat, anaesthetized with halothane. All cells received A- and C-fibre inputs and responded to innocuous and noxious stimuli applied to their excitatory receptive fields located on the extremity of the ipsilateral hindpaw. Transcutaneous application of suprathreshold (mean 3.2 T) 2 ms square-wave pulses to the centre of the receptive fields resulted in responses to A- and C-fibre activation being observed; a mean 32.4 +/- 6.0 C-fibre latency spikes were evoked per stimulus. 2 A high dose (20 micrograms) of morphine in 0.5 microliter sterile saline, microinjected into the periaqueductal grey matter (PAG) had no effect on the C-fibre-evoked activity of thirteen cells (72%) and facilitated 5 neurones (28%). Microinjection sites covered most of the PAG particularly the caudal medioventral zone. 3 A relatively high dose (6 mg kg-1, i.v.) of systemic morphine chloride, sufficient to elicit the direct spinal action of the opiate, inhibited all 5 cells tested. 4 We conclude that there is little evidence that the supraspinal action of morphine includes increased descending controls and depression of dorsal horn neurones in the rat.

Animals↗

PubMed1994

Lack of effect of microinjection of noradrenaline or medetomidine on stimulus-evoked release of substance P in the spinal cord of the cat: a study with antibody microprobes.

1. Experiments were performed on barbiturate anaesthetized, spinalized cats to investigate the effect of microinjected noradrenaline or medetomidine on the release of immunoreactive substance P in the dorsal spinal cord following peripheral nerve stimulation. The presence of immunoreactive substance P was assessed with microprobes bearing C-terminus-directed antibodies to substance P. 2. Noradrenaline or medetomidine were microinjected into the grey matter of the spinal cord, near microprobe insertion sites, at depths of 2.5, 2.0, 1.5 and 1.0 mm below the spinal cord surface with volumes of approximately 0.125 microliters and a concentration of 10(-3) M. 3. In the untreated spinal cord, electrical stimulation of the ipsilateral tibial nerve (suprathreshold for C-fibres) elicited release of immunoreactive substance P which was centred in and around lamina II. Neither noradrenaline nor medetomidine administration in the manner described produced significant alterations in this pattern of nerve stimulus-evoked release. 4. In agreement with recent ultrastructural studies these results do not support a control of substance P release by catecholamines released from sites near to the central terminals of small diameter primary afferent fibres.

Adrenergic alpha-Agonists↗

PubMed2006

Localized intradermal microinjection of tranexamic acid for treatment of melasma in Asian patients: a preliminary clinical trial.

BACKGROUND: Melasma is a common cosmetic problem among Asians. While various treatments are currently being used, there is no entirely satisfactory treatment. It was recently reported that the topical plasmin inhibitor is an effective treatment for ultraviolet-induced hyperpigmentation. OBJECTIVE: Because there are no studies assessing the efficacy and safety of localized microinjection of tranexamic acid (TA) for the treatment of melasma, we conducted a pilot study to evaluate the efficacy and side effects of this potentially new method for the treatment of melasma in Korean women. METHODS: A total of 100 women with melasma, after written consent, were enrolled for a prospective open pilot study of 12 weeks. After applying topical anesthesia, 0.05 mL TA (4 mg/mL) was injected intradermally into the melasma lesion at 1 cm intervals by using a 0.5 mL insulin syringe with a 30-gauge needle. This was repeated weekly for 12 weeks. A clinical investigator evaluated the results by using the Melasma Area and Severity Index (MASI) at baseline and at 4, 8, and 12 weeks. The patient satisfaction questionnaire was documented at 12 weeks. Safety evaluations were performed at each follow-up visit. RESULTS: Eighty-five patients completed the trial. A significant decrease in the MASI from baseline to 8 and 12 weeks was observed (13.22+/-3.02 vs 9.02+/-2.62 at week 8 and vs. 7.57+/-2.54 at week 12; p<.05 for both). The patients' self-assessment of melasma improvement was as follows: 8 of 85 patients (9.4%) rated as good (51-75% lightening), 65 patients (76.5%) as fair (26-50% lightening), and 12 patients (14.1%) as poor (0-25% lightening). Side effects were minimal and all the patients tolerated the treatment well. CONCLUSION: Based on these results, we suggest that the intralesional localized microinjection of TA acid can be used as a potentially new, effective, and safe therapeutic modality for the treatment of melasma.

Adult↗

PubMedDate not supplied

Role of the fastigial nucleus in generalized seizures as demonstrated by GABA agonist microinjections.

The cerebellum is electrically and metabolically active during seizures. Numerous studies have also shown that cerebellar electrical stimulation and lesions of the cerebellar cortex or nuclei influence seizure threshold, but there are significant contradictions, with different effects observed even in investigations using the same species and similar seizure types and experimental manipulations. Discrete intracerebral microinjection of neuroactive agents has been used to characterize the way in which other brain regions control seizures, but has not been applied to the cerebellar systems. This approach has advantages because effects are restricted to specific receptors and spare passing axons; experimental variables also can be simply specified and reproduced. We used this method to characterize the role of the cerebellar nuclei in seizures and to determine if observed effects could be reproduced with different agents at different doses. Effects of bilateral control microinjections in the fastigial (medial) cerebellar nucleus were compared with different doses of the GABAA agonist piperidine-4-sulfonic acid and the GABAB agonist (-)baclofen (Bf). Soon after injection, the animals were ataxic. After 4 min, seizures were induced by timed continuous intravenous (i.v.) bicuculline (BIC) infusion. Both GABA agonists produced significant reductions in myoclonic, clonic, and tonic seizure thresholds. Injections just dorsal or anterior to this nucleus and bilateral dentate (lateral) nucleus injections had little effect on seizures. These results demonstrate that the cerebellar system does control seizures, but does not provide support for the early concept that cerebellar stimulation and systemic phenytoin block seizures through inhibition of cerebellar nuclei secondary to Purkinje cell activation.

Animals↗

PubMed1990

Resumption of rat oocyte meiosis is paralleled by a decrease in guanosine 3',5'-cyclic monophosphate (cGMP) and is inhibited by microinjection of cGMP.

The aim of the present study was to measure the level of cyclic GMP (cGMP) compared with the level of cyclic AMP (cAMP) in rat oocytes during resumption of meiosis I (oocyte maturation) and to microinject these cyclic nucleotides into the oocyte to study their effects on oocyte maturation. Immature oocytes were obtained from prepubertal rats primed with pregnant mare's serum gonadotropin. OOcytes were isolated adn short-term cultured under conditions enabling spontaneous maturation. The levels of cGMP and cAMP decreased in the oocyte during spontaneous maturation (from 0.41 to 0.25 and from 0.64 to 0.42 fmol per oocyte respectively). The decrease was observed during the first hour of culture, and no further decline was seen after 2 h. Microinjection of cGMP or cAMP into isolated immature oocytes delayed the spontaneous maturation, cGMP being slightly more effective than cAMP, whereas 2-deoxy-cAMP (which does not stimulate protein kinase A) did not. These results demonstrate for the first time that the level of cGMP decreases in the oocyte parallel to spontaneous meiosis, as already shown for cAMP. This suggests that cGMP, as well as cAMP, may be of importance for regulating this process. This assumption is further supported by data demonstrating a delay in the maturation of oocytes injected with cGMP.

Animals↗

PubMed1986

Adenovirus type 12 E1A protein expressed in Escherichia coli is functional upon transfer by microinjection or protoplast fusion into mammalian cells.

We efficiently expressed, in Escherichia coli, and purified the protein product encoded by the human adenovirus type 12 (Ad12) 13S mRNA. The functional properties of the E1A protein were analyzed by introducing the protein by microinjection or protoplast fusion into living mammalian cells. We showed that the E. coli-expressed E1A protein induces gene expression of the adenovirus type 5 (Ad5) E1A deletion mutant Ad5dl312. The purified E1A protein rapidly and quantitatively localized to the cell nucleus after microinjection into the cytoplasm. In addition, we raised high-titered monospecific antibodies to the purified Ad12 E1A protein. Using deleted forms of an adenovirus type 2 and Ad5 hybrid (Ad2/5) E1A protein, we showed that all of the epitopes conserved between Ad2/5 E1A and Ad12 E1A protein that are recognized by the Ad12 E1A-specific antiserum map to within the first exon-encoded amino-terminal half of the protein.

Adenovirus Early Proteins↗

PubMed1990

Effect of microinjection of a low-Mr human placenta protein tyrosine phosphatase on induction of meiotic cell division in Xenopus oocytes.

Homogeneous preparations of a protein phosphatase that is specific for phosphotyrosyl residues (protein tyrosine phosphatase [PTPase] 1B) were isolated from human placenta and microinjected into Xenopus oocytes. This resulted in an increase in activity of up to 10-fold over control levels, as measured in homogenates with use of an artificial substrate (reduced carboxamidomethylated and maleylated lysozyme). Microinjected PTPase was stable for at least 18 h. It is distributed within the oocyte in a manner similar to the endogenous activity and is suggestive of an interaction with cellular structures or molecules located predominantly in the animal hemisphere. The phosphatase markedly retarded (by up to 5 h) maturation induced by insulin. This, in conjunction with the demonstration that PTPase 1B abolished insulin stimulation of an S6 peptide (RRLSSLRA) kinase concomitant with a decrease in the phosphorylation of tyrosyl residues in a protein with the same apparent Mr as the beta subunit of the insulin and insulinlike growth factor 1 receptors (M. F. Cicirelli, N. K. Tonks, C. D. Diltz, E. H. Fischer, and E. G. Krebs, submitted for publication), provides further support for an essential role of protein tyrosine phosphorylation in insulin action. Furthermore, maturation was significantly retarded even when the PTPase was injected 2 to 4 h after exposure of the cells to insulin. PTPase 1B also retarded maturation induced by progesterone and maturation-promoting factor, which presumably do not act through the insulin receptor. These data point to a second site of action of the PTPase in the pathway of meiotic cell division, downstream of the insulin receptor and following the appearance of active maturation-promoting factor.

Animals↗

PubMed1990

Induction of transformation and DNA synthesis after microinjection of raf proteins.

Full-length and N-terminal deletion mutants of human c-raf-1 cDNA were cloned into Escherichia coli expression plasmids. Bacterially expressed c-raf proteins were purified by anion-exchange, gel filtration, and affinity chromatography. Microinjection of mutant c-raf proteins into G0-arrested NIH 3T3 cells induced DNA synthesis and morphological transformation, whereas microinjection of full-length c-raf had no effect. The amino terminus of the raf protein has an important negative regulatory influence; alteration of this region resulted in increased kinase activity and oncogenicity.

Animals↗

PubMed1983

Highly purified pp60src induces the actin transformation in microinjected cells and phosphorylates selected cytoskeletal proteins in vitro.

The src gene product of Rous sarcoma virus (pp60(src)) was highly purified from a rat tumor cell line and shown to have physiological actin transformation activity in a cellular microinjection assay that measures the dissolution of actin microfilament bundles in vivo. The purified pp60(src) fraction consisted of two major proteins, seen on silver-stained sodium dodecyl sulfate-polyacrylamide gels: a 60,000-dalton (60K) protein, identified as pp60(src) by immunoprecipitation with tumor-bearing rabbit immunoglobulin G (IgG) and peptide mapping, and an unrelated 65K protein. There was no evidence for proteolytic cleavage of pp60(src). A 7,000-fold purification of the tyrosine-specific protein kinase activity of pp60(src) was achieved by this procedure. Purified pp60(src) phosphorylated tumor-bearing rabbit IgG heavy chains, casein, histones H1 and H2B, tubulin, and microtubule-associated proteins when assayed in vitro. When incubated with [gamma-(32)P]ATP in the absence of exogenous phosphoacceptor substrates, purified pp60(src) became labeled with (32)P at the tyrosine residues exclusively. Phosphatase and cyclic AMP-dependent protein kinase activities were undetectable in the purified fraction. Microinjection of highly purified pp60(src) into the cytoplasm of normal Swiss 3T3 mouse fibroblasts caused rapid and reversible dissolution of actin stress fibers, as visualized by indirect immunofluorescence with actin antibodies. The actin-disrupting activity was thermolabile and sensitive to inhibition by coinjection of tumor-bearing rabbit IgG, and purified to about the same extent (8,000-fold) as did the IgG kinase activity of pp60(src), thus implicating pp60(src) as the active agent. Examination of actin-associated proteins as substrates for the pp60(src) kinase in vitro showed that vinculin was phosphorylated directly by pp60(src), although to a small extent. Actin, myosin, and tropomyosin were not phosphorylated. Thus, pp60(src) purified by this procedure retains native functional properties and provides a useful probe for analyzing transformation-dependent changes in actin cytoarchitecture.

Actins↗