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PubMed1994

GABAB receptor-mediated mechanisms in the RVLM studied by microinjections of two GABAB receptor antagonists.

The cardiovascular effects of microinjections of two gamma-aminobutyric acid (GABA) antagonists specifically acting on GABAB receptors, 2-hydroxy-saclofen (2-OH-S) and CGP-35348, into vasopressor sites of rostral ventrolateral medulla (RVLM) were studied in rats anesthetized with urethan. Bilateral microinjection of 2-OH-S induced significant increases in mean arterial pressure (MAP) and heart rate (HR) in 21 of 26 RVLM vasopressor sites (81%); average increases obtained in the 26 sites studied were +25.2 +/- 3.0 mmHg and +12.7 +/- 2.1 beats/min. Bilateral microinjection of CGP-35348 induced significant increases in MAP and HR in 10 of 12 RVLM sites (83%). Average increases in the 12 sites studied were +27.6 +/- 3.9 mmHg and +14.6 +/- 2.5 beats/min. Sixteen rats received unilateral electrolytic lesions of one RVLM. Microinjections of either 2-OH-S or CGP-35348 into vasopressor sites within the intact RVLM significantly antagonized the depressor responses observed after injections of baclofen (20 pmol) into the same sites, whereas both GABAB antagonists did not affect the depressor response induced by microinjection of muscimol (5 pmol). These results suggest a tonic inhibitory mechanism within the RVLM mediated by GABAB receptors involved in central cardiovascular regulation.

Animals↗

PubMed2005

Attenuation of aortic baroreflex responses by microinjections of endomorphin-2 into the rostral ventrolateral medullary pressor area of the rat.

The presence of mu-opioid receptors and endomorphins has been demonstrated in the general area encompassing the rostral ventrolateral medullary pressor area (RVLM). This investigation was carried out to test the hypothesis that endomorphins in the RVLM may have a modulatory role in regulating cardiovascular function. Blood pressure and heart rate (HR) were recorded in urethane-anesthetized male Wistar rats. Unilateral microinjections of endomorphin-2 (0.0125-0.5 mmol/l) into the RVLM elicited decreases in mean arterial pressure (16-30 mmHg) and HR (12-36 beats/min), which lasted for 2-4 min. Bradycardia was not vagally mediated. The effects of endomorphin-2 were mediated via mu-opioid receptors because prior microinjections of naloxonazine (1 mmol/l) abolished these responses; the blocking effect of naloxonazine lasted for 15-20 min. Unilateral stimulations of aortic nerve for 30 s (at frequencies of 5, 10, and 25 pulses/s; each pulse 0.5 V and 1-ms duration) elicited depressor and bradycardic responses. These responses were significantly attenuated by microinjections of endomorphin-2 (0.2 and 0.4 mmol/l). The inhibitory effect of endomorphin-2 on baroreflex responses was prevented by prior microinjections of naloxonazine. Microinjections of naloxonazine alone did not affect either baseline blood pressure and HR or baroreflex responses. These results indicate that endomorphin-2 elicits depressor and bradycardic responses and inhibits baroreflex function when injected into the RVLM. These effects are consistent with the known hyperpolarizing effect of opioid peptides on RVLM neurons.

Animals↗

PubMed1984

Cardiovascular actions of microinjections of angiotensin II in the brain stem of rats.

The blood pressure and heart rate responses to microinjection of angiotensin II (ANG II) into the brain stem of urethan-anesthetized rats were studied. Microinjection of ANG II into the area postrema (AP) resulted in significant elevation of blood pressure and significant reduction of heart rate. Microinjection into the region of the nucleus tractus solitarius (NTS) yielded a significant dose-dependent elevation in blood pressure and consistent increases in heart rate. The response to microinjection of ANG II into the region of the NTS was not due to leakage into the peripheral circulation, since intravenous administration of the ANG II antagonist, saralasin, did not attenuate the response. In fact, the cardiovascular response was increased after peripheral ANG II blockade, and the heart rate, which was consistently but not significantly elevated by NTS injection alone, was significantly elevated after saralasin pretreatment. Thermal ablation of the AP did not change the heart rate or the pressor response to microinjection of ANG II into the region of the NTS, indicating that the response was not mediated through the AP.

Angiotensin II↗

PubMed1989

Baroreceptor activation or glutamate coinjection facilitates depressor responses to ANF microinjection into NTS.

As microinjection of atrial natriuretic factor (ANF) into the nucleus of the solitary tract (NTS) has been shown to elicit depressor responses [D. J. McKitrick and F. R. Calaresu. Am. J. Physiol. 255 (Regulatory Integrative Comp. Physiol. 24): R182-R187, 1988], we investigated the possibility that these responses might be facilitated either by electrical stimulation of arterial baroreceptor fibers in the aortic depressor nerve (ADN) or by simultaneous microinjection of L-glutamate (Glu) into the same sites in the NTS. Male Wistar rats (n = 51) were anesthetized with urethan (1.4 g/kg ip), artificially ventilated, and the dorsal medulla was exposed. The ADN was isolated, cut distally, and the central end was placed on bipolar stimulating electrodes. Threshold doses of 10(-7) M ANF microinjected into the NTS were combined with threshold electrical stimulation of the ADN (n = 37) or threshold doses of 0.13-0.5 M Glu (n = 14) microinjected into the NTS. There was a significant interaction between ANF microinjection and ADN stimulation in producing changes in mean arterial pressure (MAP) and heart rate [HR; P less than 0.05; -20.2 +/- 2.3 (SE) mmHg and -30.8 +/- 6.9 (SE) beats/min, respectively; n = 18]. There was also a significant interaction between ANF and Glu in producing changes in MAP and HR [P less than 0.05; -16.3 +/- 1.8 (SE) mmHg and -15.0 +/- 3.0 (SE) beats/min, respectively; n = 8]. These results indicate that ANF influences neurons in the NTS, which are also influenced by activation of arterial baroreceptors, and ANF and Glu interact in the NTS to produce facilitated cardiovascular responses.

Animals↗

PubMed1989

Intrapreoptically microdialyzed and microinjected norepinephrine evokes different thermal responses.

Norepinephrine (NE) microdialyzed into the medial preoptic area (MPO) evokes a core temperature (Tco) fall in contrast to the rise when it is microinjected. Because prostaglandin E2 (PGE2) is a contaminant of the microinjection procedure per se, we determined whether it might account for these differential thermal responses. NE (1 microgram/microliter) was bilaterally microinjected into the MPO of conscious guinea pigs treated 20 min prior with a PG synthetase inhibitor, indomethacin (Indo, 10 mg/kg, im). Under these conditions, the latency of the NE-induced Tco rise was prolonged (138 +/- 18 min). When Indo was administered both 20 min before and 20 min after NE microinjection, NE was hypothermizing. NE (10 micrograms/microliter at 2 microliters/min for 3 h) microdialyzed into the MPO, lateral septum, or anterior hypothalamus caused Tco falls, whereas it induced no Tco change when dialyzed into the lateral preoptic area, indicating site specificity. PGE2 (1 microgram/microliter) caused a Tco rise when it was dialyzed intra-MPO. Microdialysis of PGE2 and NE together neutralized each other's effects. Indo given at the end of intra-MPO NE dialysis blocked the usual recovery of Tco from its lowered value. These results indicate that NE and PGE2 in the MPO may reciprocally influence the Tco of guinea pigs. The data further suggest that PGE may account for the different responses to microinjected and microdialyzed NE.

Animals↗

PubMed1997

Adrenomedullin microinjection into the area postrema increases blood pressure.

Adrenomedullin (ADM) circulates in the blood at concentrations comparable to other vasoactive peptides with established roles in cardiovascular regulation. Intravenously administered ADM produces a clear hypotensive effect, whereas intracerebroventricular microinjections result in increases in blood pressure (BP). Recently, we demonstrated that ADM influences neurons of the area postrema (AP), a central nervous system site implicated in cardiovascular control. However, to address directly the physiological significance of the actions of ADM at the AP, an in vivo microinjection study was undertaken. ADM, at two concentrations (1 and 10 microM), in volumes of 50, 100, and 200 nl, was microinjected into the AP or NTS of 21 urethan-anesthetized male Sprague-Dawley rats. Microinjection of 10 microM ADM (100 nl) resulted in significant transient (2-5 min) increases in BP [120 s area under the curve (AUC): 684.3 +/- 268.6 mmHg/s (P < 0.05)], and heart rate (HR) [AUC: 12.5 +/- 4.5 beats/min (P < 0.05)]. The lower concentration of ADM (1 microM) had no effect on either BP (179.1 +/- 143.6 mmHg/s) or HR (0.8 +/- 2.6 beats/min). ADM was also microinjected into the immediately adjacent nucleus of the solitary tract, where it was found to be without effect on either BP or HR. This study demonstrates, for the first time, a physiological role for ADM acting at a specific brain site, the AP, to produce significant cardiovascular responses.

Adrenomedullin↗

PubMed1999

Respiratory responses to thyrotropin-releasing hormone microinjected into the rabbit medulla oblongata.

We investigated the respiratory role of thyrotropin-releasing hormone (TRH) input to medullary structures involved in the control of breathing in anesthetized, vagotomized, paralyzed, and artificially ventilated rabbits. Microinjections (10-20 nl) of 1 or 10 mM TRH were performed in different regions of the ventral respiratory group (VRG), namely the rostral expiratory portion or Bötzinger complex (Böt. c.), the inspiratory portion, the transition zone between these two neuronal pools, and the caudal expiratory component. TRH microinjections were also performed in the dorsal respiratory group (DRG) and the area postrema (AP). Injection sites were localized by using stereotaxic coordinates and extracellular recordings of neuronal activity; their locations were confirmed by subsequent histological control. TRH microinjections in the Böt. c. and the directly caudally located region where a mix of inspiratory and expiratory neurons were encountered elicited depressant respiratory responses. TRH microinjections were completely ineffective at sites within the inspiratory and the caudal expiratory components of the VRG. TRH microinjections in either the DRG or the AP induced excitatory effects on inspiratory activity. The results show for the first time that TRH may exert inhibitory influences on respiration at medullary levels by acting on rostral expiratory neurons and that not only the DRG, as previously suggested, but also the AP may mediate TRH-induced excitatory effects on respiration.

Animals↗

PubMed1988

Cytochemical analysis of the reconstitution of endoplasmic reticulum after microinjection of rat liver microsomes into Xenopus oocytes.

Fragments of rough and smooth endoplasmic reticulum purified from rat liver were injected into Xenopus oocyte cytoplasm. Light and electron microscopy, cytochemistry, immunocytochemistry, and enzyme assay were employed to determine the fate of heterologous membranes in the host cytoplasm. The in vivo-incubated microsomes disappeared in a time-dependent manner. Within 3 hr, rough microsomes were replaced by flattened ER cisternae and smooth microsomes were replaced by a network of anastomosing tubules. Polyclonal antibodies against rat liver microsomes and protein A-gold complexes were applied to glycol methacrylate sections of microinjected oocytes. Specific labeling was observed over discrete rough and smooth ER cisternae 3 hr after microinjection. Endogenous ER was not labeled by this technique, and label was not observed when sections were treated with pre-immune antibodies. Diaminobenzidene cytochemistry of microinjected rat lacrimal gland microsomes revealed enzyme activity in heterologous microsomes after 3 hr of in vivo incubation. Control injected microsomes (inactivated by heat denaturation) became associated with autophagic vacuoles, coincident with changes in lysosomal activity. Freshly isolated un-denatured microsomes did not provoke changes in lysosomal activity, and glucose-6-phosphatase activity associated with microinjected membranes could be detected 21 hr after in vivo incubation. Since rat liver microsomes reconstitute after in vivo incubation into cytoplasmic structures resembling those from which they were derived, we conclude that the microinjected membrane fragments act as templates for their own three-dimensional organization.

Animals↗

PubMed2003

Nifedipine potentiates the antinociceptive effect of endomorphin-1 microinjected into the periaqueductal gray in rats.

UNLABELLED: Endomorphin-1 is a novel endogenous mu-opioid ligand. We investigated the antinociceptive interaction between endomorphin-1 and nifedipine, an L-type calcium channel blocker, microinjected into the midbrain ventrolateral periaqueductal gray (vPAG), using the spinally-organized tail-flick test and the supraspinally-organized tail-pressure test in rats. Sprague-Dawley rats were stereotaxically implanted with a guide cannula lowered into the vPAG. Microinjection of endomorphin-1 into the vPAG led to dose-related increases in antinociceptive responses in the tail-flick test and tail-pressure test. Pretreatment with the mu-opioid receptor-selective antagonist beta-funaltrexamine blocked the antinociceptive effect of endomorphin-1. Pretreatment with beta-funaltrexamine alone had no effect on the tail-flick latency and tail-pressure threshold. Microinjection of nifedipine alone into the vPAG did not produce an antinociceptive response in the tail-flick test and tail-pressure test. However, injection of nifedipine into the vPAG potentiated the antinociceptive effect of endomorphin-1, producing a significant leftward shift in the dose-response curve of endomorphin-1 in both the tail-flick and tail-pressure tests. This result shows that the potent antinociceptive effect of endomorphin-1 microinjected into the vPAG is mediated through the mu-opioid receptor and is potentiated by concomitant administration of nifedipine. IMPLICATIONS: This study shows that the potent antinociceptive effect of endomorphin-1 microinjected into the ventrolateral periaqueductal gray is potentiated by concomitant administration of nifedipine. This suggests that calcium channel blockers may enhance the analgesia of opioids in patients with calcium channel blocker treatment.

Analgesics, Opioid↗

PubMed1986

Antidiuretic effects of dibutyryl-cyclic AMP microinjected into the hypothalamic paraventricular nucleus in a water-loaded and ethanol-anesthetized rat.

Effects of dibutyryl-cyclic AMP (db-cAMP) and cyclic AMP (cAMP) when microinjected into the hypothalamic paraventricular nucleus (PVN) in a water-loaded and ethanol-anesthetized rat on the rate of urine outflow, urine osmotic pressure and other visceral functions were investigated. The microinjection of db-cAMP decreased the rate of urine outflow with concomitant increase in the urine osmotic pressure, but did not change mean blood pressure, heart rate, respiration rate and rectal temperature. The antidiuretic effect of db-cAMP was more potent than the effect of cAMP, the median effective doses (ED50) being approx. 40 nmol for db-cAMP and more than 300 nmol for cAMP, respectively. The time-courses for the antidiuretic effects and for the increase in the urine osmotic pressure showed a similar pattern, with the maximal effect at approx. 30 to 40 min and the duration of approx. one hour or longer. The effect of db-cAMP was potentiated by pretreatment with methylxanthines and inhibited by pretreatment with atropine. A second microinjection of db-cAMP induced a less potent antidiuretic effect than the first microinjection (tachyphylaxis). The results indicated the antidiuretic effects of microinjection of db-cAMP and cAMP into the PVN, and a possible mechanism for this was discussed.

1-Methyl-3-isobutylxanthine↗

PubMed1994

Effects of time of deoxyribonucleic acid microinjection on gene detection and in vitro development of bovine embryos.

In vivo fertilized embryos were surgically collected from superovulated dairy cows to evaluate microinjection on embryo development and utilized the polymerase chain reaction technique for selection of transgenic embryos. Seventy-two percent of the embryos with visible pronuclei or nuclei were microinjected with DNA, and the remaining 28% served as uninjected controls. All embryos were cocultured with bovine oviductal epithelial cells. Mean final development scores of embryos within the same initial cell stage at collection were unaffected by microinjection. After 144 h of culture, 45% of the microinjected embryos developed to the morula or blastocyst stage. The transgene was detected in 50, 10, and 9% of demimorulae from embryos microinjected at the 1-, 2-, and 4-cell stages. Frequency of transgene detection was higher in morulae from 1-cell embryos than in morulae from 2- and 4-cell embryos. Use of in vitro coculture, embryo bisection, and polymerase chain reaction technique facilitated selection of bovine embryos that carried the transgene.

Animals↗

PubMed1995

Nuclear transfer in the bovine using microinjected donor embryos: assessment of development and deoxyribonucleic acid detection frequency.

Bovine embryos that had been microinjected with DNA were examined for their potential use as donor embryos in nuclear transfer. Donor embryos were obtained from oocytes collected by transvaginal oocyte aspiration, matured and fertilized in vitro, microinjected with a murine whey acidic protein-human protein C genomic DNA construct, and cultured in vitro on liver cells of buffalo rat (Rattus norvegicus). Blastomeres from these embryos were transferred into enucleated bovine oocytes received from an abattoir by electrofusion at 40 h postmaturation. Following 7 d of culture, the developmental stage was recorded, and resulting embryos were prepared for analysis by polymerase chain reaction. Embryos that were derived from microinjected donor embryos did not differ from control donor embryos (11 vs. 8.6%) in development to the morula and blastocyst stage. Of the biopsies from 20 microinjected donor embryos, 19 were positive for the injected DNA. Of 37 embryos developing normally, only 12 (32.4%) were positive for the injected DNA. These results indicate that microinjected embryos can be successfully used in a nuclear transfer program to produce additional viable embryos and that these embryos may be reliably screened for the transgene for transfer to recipients.

Animals↗

PubMed1999

Bovine follicular dynamics, oocyte recovery, and development of oocytes microinjected with a green fluorescent protein construct.

The present study was carried out to 1) evaluate the viability of in vitro fertilized zygotes after microinjection of DNA, 2) assess the influence of oocyte quality upon the development rate of embryos when injected with DNA, and 3) determine the integration frequency of green fluorescent protein DNA into microinjected embryos. Oocytes were aspirated from ovaries of nine nonlactating Holsteins and were categorized into grades A, B, C, and D. At 16 h after in vitro fertilization, approximately half of the pronuclear stage presumptive zygotes were classified as having 1 pronucleus or 2 pronuclei, and they were microinjected with DNA constructs. A potential predictor of DNA integration frequency at d 10 was assessment of the incidence of green fluorescing embryos. The proportion of cleaved embryos that developed to morulae or blastocysts was not different between groups with 1 pronucleus injected (45%), 1 pronucleus uninjected (64%), or 2 pronuclei injected (49%). However, the development of morulae or blastocysts was higher in the group with 2 pronuclei uninjected (69%). The overall developmental score of green fluorescent protein-positive embryos was higher for grade A oocytes (1.3 +/- 0.1) than for grade B (0.8 +/- 0.1), C (0.6 +/- 0.1), or D (0.3 +/- 0.1) oocytes. The results show that production of transgenic bovine blastocysts can occur from the microinjection of a presumptive zygote having only one visible pronucleus. Initial oocyte quality is an important factor in selection of oocytes suitable for microinjection of DNA and for preimplantation development to produce bovine transgenic embryos.

Animals↗

PubMed1976

Microinjection of tRNA into amphibian oocytes.

The microinjection technique affords us the possibility to introduce purified components into living cells and to answer the question of what effects the change introduced has on cellular metabolism. This technique can therefore be used to test the hypothesis that transfer RNA plays a regulatory role in cellular protein synthesis. Prior to these experiments it is important, however, to test whether transfer RNA microinjected into amphibian oocytes is stable and functional inside this cells. These two questions are answered affirmatively in this report. The stability of tRNA was tested by following the content of TCA precipitable counts inside the oocytes at different times after microinjection of radioactive yeast and E. coli tRNA and by polyacrilamide gel electrophoresis of the material recovered from the cell. The results clearly indicate that tRNAs are resistant to the action of occyte ribonucleases that degrade other RNAs such as 5S RNA. The functionality of the injected tRNA was tested by assaying the intracellular aminoacylation of microinjected yeast tRNA. The aminoacylation of bulk yeast (3H) tRNA introduced into Xenopus laevis oocytes was tested by the capacity of the material recovered 5 hours after injection into the cell to form a ternary complex with wheat protein synthesis elongation factor 1 and GTP. The complex only forms with aminoacyl-tRNA and not with unacylated tRNA. This method showed that at least 80% of the tRNA introduced into the cell was aminoacylated in vivo. A direct assay for internal aminoacylation made use of microinjection of pure tRNAPhe and subsequent determination by phenol extraction of (14C)Phe-tRNA content of oocytes that had been incubated for 2 hours in a medium containing (14C)phenylalanine. The results obtained showed that the oocytes could internally aminoacylate 200-500 times more tRNAPhe that the cell normally contains. Appropiate controls demonstrated that the aminoacylation was aminoacid and tRNA specific and that periodate oxidized tRNAPhe could not be in vivo aminoacylated but tRNAPhe deprived of its Y base could accept the aminoacid. A brief study demonstrated that bulk yeast tRNA and tRNAPhe without its Y base did not inhibit endogenous protein synthesis but a similar amount of tRNAPhe caused 50% inhibition and periodate-oxidized tRNAPhe a 95% inhibition.

Amphibians↗

PubMed2002

[Nerve tissue morphological study of tutin microinjection into pontine NPBM in two hours].

OBJECTIVE: To observe whether Tutin microinjection into the pontine NPBM respiratory area of rabbit will cause morphological damage to that area two hours later. METHODS: At two hours after the microinjection of Tutin into NPBM, the experimental effects on respiration came to be remarkable and the physiological condition was well, the rabbit was subjected to morphological sampling then. The sample was cut into slices for LM (Nissl dyeing) and transmission EM observation and photography. RESULTS: Under the LM and EM examination, no remarkable morphological damage done by Tutin microinjection into the pontine NPBM was observed. By comparing the Tutin-injected side with the other side of NPBM where equal normal saline microinjection was given, no apparent morphological difference could be found. CONCLUSION: In our experiment condition, there was no morphological damage caused by Tutin microinjection into pontine NPBM respiratory area of the rabbit.

Animals↗

PubMed2003

Trans-species transfer of Wolbachia: microinjection of Wolbachia from litomosoides sigmodontis into Acanthocheilonema viteae.

Intracellular bacteria of the genus Wolbachia are found in most filarial nematodes, but are lacking in some species like Acanthocheilonema viteae. Due to their symbiotic nature and their role in the pathology of filarial infections they are considered to be potential targets for intervention against filarial infections in man. Infection of A. viteae (a species which does not naturally carry Wolbachia) with Wolbachia bacteria could allow comparative studies on the effect of the endobacterium on the parasite and on the host's immune systems. As a step towards such studies we microinjected adult female A. viteae with Wolbachia obtained from Litomosoides sigmodontis. The bacteria were isolated from L. sigmodontis by density-gradient centrifugation, microinjected into A. viteae worms and bacterial DNA detected by PCR with Wolbachia specific primers (ftsZ gene). Microinjected worms were cultured in vitro, and 81% survived for 10 days. Implantation of microinjected worms into Meriones unguiculatus, the rodent host of A. viteae resulted in 38% survival. The DNA of the microinjected worms recovered from jirds 8 weeks after implantation contained Wolbachia DNA as shown by PCR, suggesting that Wolbachia of L. sigmodontis can be horizontally transmitted to A. viteae.

Animals↗

PubMed1987

Differential distribution of cytokeratins after microinjection of anti-cytokeratin monoclonal antibodies.

In order to investigate the relationship of different cytokeratins within one cell, monoclonal antibodies directed against three trophectoderm cytokeratins TROMA 1, 2 and 3 were microinjected into mouse teratocarcinoma-derived trophoblastoma cells and indirect immunofluorescence tests were used to follow the subsequent localization of their respective antigens Endo A, B and C. Microinjection of TROMA 1 or 2 resulted in the perinuclear collapse of Endo A, B and C-containing filaments. Microinjection of TROMA 3 resulted in the perinuclear collapse of filaments containing Endo A and B, whereas Endo C condensed into cytoplasmic aggregates which appear as speckles in the fluorescence microscope. The speckles were electron microscopically located using indirect gold-labeling techniques and had a dense, granulous structure. They were often found to be associated with microtubules, although colchicine treatment before microinjection did not interfere with speckle formation. These experiments demonstrate that cytokeratins can become differentially distributed within the cytoplasm after microinjection of an anti-cytokeratin monoclonal antibody. Since Endo A is a type II cytokeratin and Endo B and C are type I cytokeratins, these results suggest that different members of one cytokeratin subfamily may be associated with cytokeratin filaments which have different functions within the same cell.

Animals↗

PubMed1988

Expression of thermotolerance following microinjection of glutathione disulfide.

Asynchronous Chinese hamster ovary cells were microinjected with glutathione disulfide (GSSG). Successfully injected cells were scored by coinjecting FITC-dextran with GSSG, followed by fluorescent microscopy. After microinjection, cells were incubated for 2.5 h at 37 degrees C to permit thermotolerance development and then heated at 45 degrees C for 40 min. Cellular heat sensitivity was quantitated by counting the number of grains per cell after labeling heated cells with tritiated amino acids and processing for autoradiography. The data show that microinjection of GSSG induced thermotolerance which increased the number of grains per cell up to 500% of controls. Cells that were exposed to similar concentrations of GSSG in culture medium without microinjection or microinjected without GSSG did not develop thermotolerance.

Animals↗