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Results for “Microinjection”

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PubMed1991

After microinjection hemimethylated DNA is converted into symmetrically methylated DNA before DNA replication.

In this investigation we analysed the maintenance methylation activity of the mammalian cell DNA methyltransferase by microinjection of hemimethylated HSV-tk DNA into thymidine kinase-negative rat 2 cells. We found that the hemimethylated DNA was efficiently converted into symmetrical methylated molecules before DNA replication. Furthermore, integration of the trans-DNA into the host genome is an early event after gene transfer.

Animals↗

PubMed1996

In vitro synthesized SV40 cRNA is trans-spliced after microinjection into the nuclei of mammalian cells.

We present for the first time experimental evidence that in vitro synthesized RNA (cRNA) is trans-spliced after microinjection into the nuclei of mammalian tissue culture cells. The template used for cRNA synthesis was the early SV40 BstXl/BamHI DNA fragment. This DNA fragment encodes exclusively for the second T-antigen exon and contains the intact small t-antigen intron. To generate the corresponding mRNA (T1-mRNA) by trans-splicing, the cells utilize a 5' cryptic splice site located within the second T-antigen exon of one cRNA molecule which is spliced to the small t-antigen 3' splice site of another cRNA molecule. Formation of the T1-mRNA by trans-splicing was confirmed by RT-PCR analysis and DNA sequencing. Efficient trans-splicing required that competitive small t-antigen cis-splicing be inhibited by deletion of the small t-antigen 5' splice site. The T1-mRNA was not generated when the cryptic 5' splice site was mutated.

Animals↗

PubMed1987

Activation of cryptic promoters of human c-myc genes in microinjected Xenopus laevis oocytes.

The human cellular oncogene c-myc contains two promoters at the 5' end of its first non-coding exon. Cryptic promoters located within the first intron are also activated to synthesize aberrant c-myc mRNAs in some Burkitt lymphomas with a t(8: 14) chromosome translocation in which a part of the gene structure, often the 5' non-coding exon, is truncated. We have shown elsewhere that microinjected plasmid DNA carrying a normal, intact human c-myc gene directs efficient faithful transcription from its own two promoters in Xenopus laevis oocytes. Here, I have investigated the expression of different recombinants carrying various constructs of the c-myc gene in frog oocytes in order to understand the activation mechanism of those cryptic promoters. Aberrant c-myc transcripts initiating from cryptic promoters within the first intron are undetectable when the intact or truncated c-myc gene construct is used. However, the cryptic promoters can be activated in Xenopus oocytes if the truncated c-myc gene construct is fused with simian virus 40 sequences containing a 21 base-pair repeat and the replication origin. Xenopus oocytes will be useful for further investigation of enhancing elements involved in the translocated and activated c-myc genes in Burkitt lymphoma cells.

Animals↗

PubMed1982

Inhibition of pulsatile luteinizing hormone release by morphine microinjected into mesencephalic dorsal raphe.

Blood samples were collected via jugular catheters from ovariectomized rats at 10-minute intervals for one hour before and two hours after microinjection of -.5 microliter of either saline vehicle or morphine sulfate (10 micrograms) into the dorsal raphe nucleus (DRN) or adjacent periaqueductal gray by means of chronically-implanted guide cannulae. LH was measured by radioimmunoassay and mean pre-injection and mean post-injection values were compared for each rat (t test) as well as for each treatment group (paired t test). Neither saline in DRN nor morphine at other sites significantly altered circulating LH. A significant decrease in LH was observed following injection of morphine into DRN. This effect of morphine was prevented by pre treatment of the animals with the narcotic antagonist naltrexone (10 mg/kg i.v.), indicating the involvement of opiate receptors. These results indicate that DRN is one site at which systemically-administered morphine might act, and suggest the possibility of participation of this mechanism in modulation of LH release by endogenous opioids.

Animals↗

PubMed1983

The blood pressure changes resulting from the microinjection of fragments of the beta endorphin molecule into the nucleus tractus solitarii (NTS).

Unilateral microinjection of gamma endorphin (1-17) into the nucleus tractus solitarii (NTS) of the urethane anaesthetised rat produces a dose-related pressor response. This effect has been examined in the present study, by means of fragments of the molecule. Unilateral application of the peptides 6-16, 10-16, 6-17 and 10-17 (1 ng) all produced an equivalent rise in blood pressure, however the time course of 6-16 and 10-16 was more rapid than that of gamma endorphin or the fragments containing the 17th amino acid. Naloxone (10 ng) administered into the NTS, did not affect the maximum rise in pressure induced by 6-17 (1 ng). It is concluded that in addition to a depressor region, which is dependent on the N-terminal tyrosine, the beta endorphin molecule (1-31) also has a pressor region, which is present within the amino acid sequence 10-16, and is a non-opiate effect.

Animals↗

PubMed1986

Aphagia in the rat following microinjection of neurotensin into the ventral tegmental area.

Neurotensin was microinjected into the lateral cerebral ventricle and the ventral tegmental area of rats which had been deprived of food for 18 hours. Both routes of administration resulted in a significant reduction of food intake compared to vehicle control injections. Additionally, the dose of neurotensin required to produce aphagia following ventral tegmental injection was substantially less than the dose required by the ventricular route. The results are discussed in relation to a possible site and mode of action for this neuropeptide.

Animals↗

PubMed1988

Feeding elicited by dynorphin (1-13) microinjections into the ventral tegmental area in rats.

Both the endogenous opioid peptide, dynorphin (1-13) (DYN), and morphine elicited dose-dependent feeding when microinjected into the ventral tegmental area of food-satiated rats. DYN was 50,000 times more potent than morphine in producing feeding. Whereas the ED50 for morphine was in the nanomole range, the ED50 for DYN was in the femtomole range. Administration of a narcotic antagonist attenuated DYN-elicited feeding. These data suggest a possible role for DYN in the VTA in opioid modulation of feeding behavior.

Animals↗

PubMed1988

Comparative studies of the ingestive behaviors produced by microinjections of muscimol into the midbrain raphe nuclei of the ventral tegmental area of the rat.

Microinjection of the GABA-A agonist muscimol into the median (MR) or dorsal (DR) raphe nuclei or the ventral tegmental area (VTA) of non-deprived rats induced intense feeding and drinking in a dose-dependent and site-specific manner. Lower doses of muscimol were required to increase food intake, spillage and water intake with injections into the MR than with injections into the other two sites. These data demonstrate that the MR is a more sensitive site for the elicitation of ingestive behavior than either the DR or the VTA.

Animals↗

PubMed1992

Similarities between the akinesia induced by carbachol microinjections into the pontine reticular formation and neuroleptic catalepsy.

We reported previously that microinjections of carbachol directly into the pontine reticular formation of rats induced intense akinesia. In the present article we report results of tests for rigidity, righting, bracing and clinging which were conducted with the purpose to characterize behaviorally this type of akinesia. After injections of 5-15 micrograms/0.5 microliter of carbachol into the pontine reticular formation the rats were cataleptic, were not rigid when equilibrium was not challenged, had strong righting reflexes and strong bracing and clinging responses. This type of akinesia is different from the catatonia induced by systemic morphine (20 mg/kg IP), but similar to the catalepsy induced by systemic injections of haloperidol (5 mg/kg IP). It is thus suggested that the cataleptic state produced by topical carbachol in the pons is related to the dopaminergic mechanisms important for the cataleptic effect of the neuroleptic drugs.

Analysis of Variance↗

PubMedDate not supplied

Microinjection of Micrococcus luteus UV-endonuclease restores UV-induced unscheduled DNA synthesis in cells of 9 xeroderma pigmentosum complementation groups.

The UV-induced unscheduled DNA synthesis (UDS) in cultured cells of excision-deficient xeroderma pigmentosum (XP) complementation groups A through I was assayed after injection of Micrococcus luteus UV-endonuclease using glass microneedles. In all complementation groups a restoration of the UV-induced UDS, in some cells to the repair-proficient human level, was observed. Another prokaryotic DNA-repair enzyme, T4 endonuclease V, restored the UV-induced UDS in a similar way after microinjection into XP cells. Since both enzymes specifically catalyse only the incision of UV-irradiated DNA, we conclude that this activity is impaired in cells of all 9 excision-deficient XP complementation groups tested.

Cells, Cultured↗

PubMed1982

Blood pressure and heart rate responses to microinjection of vasopressin into the nucleus tractus solitarius region of the rat.

The nucleus tractus solitarius (NTS) region in the rat has been shown to receive arginine vasopressin (AVP) and oxytocin (OT) neurophysin-containing neuronal projections from the suprachiasmatic (SNC) and paraventricular nucleus (PVN). Thus, vasopressin and oxytocin might have central influences on the circulation. This as investigated by measuring arterial blood pressure and heart rate (HR) responses following microinjection of vasopressin and oxytocin (0.1, 1.0 and 10.0 ng) into the right nucleus tractus solitarius region of rats anesthetized with urethane. Injections of vasopressin into nucleus tractus solitarius produced dose-related increases in blood pressure and heart rate. The effect of oxytocin on the blood pressure and heart rate was of a lesser magnitude without showing a dose-response relationship. Equivolumetric injections of vehicle and luteinizing hormone-releasing hormone (LH-RH) peptide had no detectable effect on blood pressure and minimal effect on heart rate. Injections of vasopressin into three different sites in the brain stem (1 mm anterior, posterior, and lateral to the tractus solitarius) did not produce significant hemodynamic changes. Intravenously injected vasopressin produced increments in blood pressure only at the highest dose level (10.0 ng) and a decrease rather than an increase in heart rate. Ganglionic blockade significantly reduced pressor responses to vasopressin injected into the nucleus tractus solitarius region and completely eliminated HR responses. Pretreatment of the nucleus tractus solitarius with a vasopressin antagonist abolished the blood pressure and heart rate responses produced by injection of vasopressin. These results suggest that vasopressin acts in the region of the nucleus tractus solitarius to exert a central action on the circulation.

Animals↗

PubMed1983

Vasopressor and depressor areas in the rat medulla. Identification by microinjection of L-glutamate.

L-Glutamate (in 0.1 microliter of 0.9% NaCl) was injected via multibarrelled glass micropipettes into the ventrolateral medulla of urethane-anesthetized rats. This area, explored stereotaxically, extended from 2.2 mm rostral (near trapezoid bodies) to 0.4 mm caudal, with respect to the obex, 2.4 mm lateral to the mid-line on each side and 2.4 mm deep from the ventral surface of the medulla. Two types of responses were elicited by injection of L-glutamate. One type was a dose-related increase in arterial pressure and heart rate; these responses were elicited from the lateral portion of nucleus reticularis gigantocellularis, the medial aspect of nucleus reticularis parvocellularis and the dorsal-lateral reticular nucleus. The second type of response was a dose-related fall in arterial pressure with no change in heart rate; this response was localized caudal to pressure areas in the caudal ventrolateral part of nucleus reticularis gigantocellularis, ventrolateral nucleus reticularis ventralis, nucleus ambiguous and the A1 region. Glutamic acid diethylester (GDEE), an antagonist of L-glutamate, blocked all the cardiovascular effects of L-glutamate. These results indicate the presence of receptors for glutamate in the pressor and depressor areas. Glutamic acid diethylester caused a fall in blood pressure when injected on its own into pressor sites suggesting the existence of a glutaminergic input to the pressor sites. Inhibition of neuronal activity in pressor sites produced by microinjection of muscimol (a potent neuroinhibitory analogue of GABA) caused a decrease in blood pressure. On the other hand, pressor responses resulted following similar inhibition in the depressor sites. These results indicate that the pressor and depressor sites identified in the ventral medulla of the rat may have an important role to play in central cardiovascular regulation.

Animals↗

PubMed1990

Intranigral microinjection of neurotensin suppresses feeding in food deprived rats.

This study was conducted to determine whether the substantia nigra is involved in the anorectic effect of centrally administered neurotensin. Microinjection of neurotensin (2.5 - 10.0 micrograms) into the substantia nigra produced a significant suppression of feeding in 18 hour food deprived rats. To determine if the anorectic effect resulted from a general impairment of sensory-motor functioning, eight behaviors were measured in a separate study. Significant effects were found in only two of the eight behavioral categories (sniffing and eating), and only at the highest neurotensin dose (10.0 micrograms). These results suggest that the substantia nigra may be involved in the anorectic effect of neurotensin.

Animals↗

PubMed1977

Integrated excitatory response to microinjections of L-glutamic acid into the reticular system of the toad.

Microinjections of L-glutamic acid into the mesencephalic tegmentum and diencephalon of anesthetized toads evoked an excitatory response consisting of electrocephalographic and electromyographic activation, and a rise in arterial blood pressure. Electromyographic responses were reduced or absent in diencephalically injected toads. Except for this case, all other components of the response always appeared together and presented the same time course. L-glutamic acid was able to activate a group of neurons at different levels of the reticular system involved in an integrated response of ergotropic nature.

Animals↗

PubMed1982

Effects of histamine microinjections into the hippocampus on open-field behavior in rats.

Increasing evidence supports the concept that histamine would subserve neurotransmitter functions in the brain. In hippocampus, some cases of histamine receptors coupled to adenylate cyclase has been described. In our laboratory we were interested in studying the possible physiological role of these histamine receptors and behavior. Unilateral cannulae were stereotaxically implanted in the caudal hippocampus in adult male rats. Forty-eight hours later, after an acute microinjection of histamine (1 microgram), 3-methyl-histamine (1 microgram) or saline (1 microliter), the following behavioral parameters were measured by an observer in an open-field: locomotor, rearing, sniffing, grooming, social sniffing and holding activities. It was observed that histamine inhibited locomotor and rearing activities; whereas, 3-methyl-histamine induced an increase in almost all behaviors. Results support the concept of a probable function of histamine in exploratory behavior in the rat.

Animals↗

PubMed1985

Some behavioral effects of microinjections of noradrenaline and serotonin into the amygdaloid body of the rat brain.

The effects of microinjections of noradrenaline (NA) and serotonin (5HT) into the basomedial part of the amygdaloid body (BM AB) on various forms of behavior were studied in rats. NA as well as 5HT administered to the BM AB had dose-related and general inhibitory influence on the rats' behavior in the open field test. The reactivity of rats to pain (tail compression) was attenuated by 5HT injections. The effect of NA in this test was less clear, though some inhibitory tendency was also present. Pretrial injections of NA (40 micrograms) and 5HT (40 micrograms) significantly impaired the retention of a passive avoidance reaction. NA injections also produced some disinhibitory effects on shock-suppressed drinking in the conflict test. The effects of intra-amygdalar administration of NA and 5HT on open field behavior were potentiated by pretreatment of rats with nialamide, a monoaminooxidase inhibitor. The results are discussed in terms of an involvement of the amydalar NA and 5HT in the regulation of animal behavior.

Amygdala↗

PubMed1994

Reduction in food and water intake induced by microinjection of interleukin-1 beta in the ventromedial hypothalamus of the rat.

Interleukin-1 (IL-1) is a cytokine which is released during immune activation and mediates some of the host's responses to infection and inflammation. Increasing evidence suggests that it also has a role as an intrinsic neuromodulator in the central nervous system. We report here that microinjections of 5 and 30 ng (286 fmol and 1.71 pmol) of recombinant human IL-1 beta in the ventromedial nucleus of the hypothalamus (VMH) of adult male rats time- and dose-dependently induce anorexia and weight loss in two experimental paradigms: rats allowed free-access to food and water and food-restricted rats trained to press a lever for food on a fixed ratio 10 schedule. IL-1 beta (5 ng) diminished food and water consumption by 45 and 30%, respectively, and decreased body weight for at least 24 h postinjection in rats fed ad lib. These effects were more severe and lasted at least 48 h after infusion of the larger dose of 30 ng. The IL-1 beta-induced anorexia and weight loss were neither as large nor as long-lasting in food-restricted rats. Operant responding for food was decreased 2-4 h postinfusion of 5 ng IL-1 beta and 2-8 h after 30 ng IL-1 beta, but in both cases returned to baseline within 24 h. Body weight was decreased compared to saline injections from 4 to 24 h postinfusion. Nevertheless, when allowed to eat ad lib for the 24 h immediately following the behavioral testing, body weight returned to control (5 ng) or near control levels (30 ng).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1989

Microinjection of cultured rat embryos: a new technique for studies in chemical dysmorphogenesis.

The utility of a new technique for exposure of cultured whole rat embryos to potential dysmorphogens was demonstrated with nitrosofluorene (NF), a cytotoxic and mutagenic metabolite of 2-acetylaminofluorene (AAF). At an initial concentration in the culture medium of 41 microM, NF produced a 100% incidence of defects in axial rotation with no significant effect on prosencephalic development, consistent with previous reports. This route of exposure was also associated with a significant decrease in yolk sac vasculature and protein content. However, when 2 to 20 ng of NF was microinjected directly into the amniotic space, the predominant malformation observed was prosencephalic hypoplasia. Injection of 10 ng NF resulted in approximately equivalent decreases in viability as 41 microM NF dissolved in the culture medium, but produced only a 41% incidence of rotation defects and a 27% incidence of open neural tubes in the rhombencephalic region. The protein content of injected conceptuses was significantly reduced in the embryo, but not in the visceral yolk sac. When 10 ng of NF was injected inside the yolk sac but outside the amnion, the incidence of abnormal rotation was increased to 75%, and the severity of prosencephalic hypoplasia as well as the incidence of neural tube abnormalities was attenuated. The protein content of both the embryo and yolk sac was significantly decreased relative to that of the controls. The data are consistent with the suggestion that NF elicits defects in axial rotation primarily via its effects on the visceral yolk sac and demonstrate the capacity of this technique to provide insights into mechanistic aspects of chemical dysmorphogenesis.

Animals↗